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PRODUCT INFORMATION AND MANUAL FlowCytomix Human Th1/Th2 11plex Sample Kit (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α, TNF-β) BMS810FFSA For research use only. Not for diagnostic or therapeutic procedures. 26 Tests (11 analytes) Human Th1/Th2 11plex BMS810FFSA Bender MedSystems GmbH Campus Vienna Biocenter 2 A-1030 Vienna, Austria, Europe TABLE OF CONTENTS 1 Intended Use 3 2 Summary 4 3 Principles of the Test 5 4 Reagents Provided 7 5 Storage Instructions 10 6 Specimen Collection 10 7 Materials Required But Not Provided 11 8 Precautions for Use 12 9 Preparation of Reagents and Samples 14 10 Technical Tipps 18 11 Test Protocol 19 12 Cytometer Setup 24 13 Calculation of Results 33 14 Limitations 35 15 Performance Characteristics 36 16 FAQ for FlowCytomix Kits 40 17 Bibliography 44 18 Ordering Information 46 19 Reagent Preparation Summary 47 20 Test Protocol Summary 48 BMS810FFSA human Th1/Th2 11plex 27.05.09 (05) 3 1 INTENDED USE BMS810FFSA is a bead based Analyte Detection System for quantitative detection of human Interferon-γ, Interleukin-1β, Interleukin-2, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-8, Interleukin-10, Interleukin-12p70, Tumor Necrosis Factor α and Tumor Necrosis Factor β by Flow Cytometry. BMS810FFSA is for research use only. Not for use in diagnostic or therapeutic procedures. BMS810FFSA human Th1/Th2 11plex 4 2 SUMMARY The term TH1 cytokines (referred to also as Type-1 cytokines) and TH2 cytokines (referred to also as Type-2 cytokines) refers to the patterns of cytokines secreted by two different subpopulations of murine CD4 (+) Tcells that determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Numerous cells other than T-cells expressing CD4 have been shown to be capable of producing TH1 cytokines and TH2 cytokines. These cells include CD8 (+) T-cells, monocytes, natural killer cells, B-cells, eosinophils, mast cells, basophils, and other cells. Type-1 cytokines include IL-2, IFNγ, IL-12 and TNFβ, while Type-2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13. Type-1 helper cells (TH1), but not type-2 helper cells (TH2), secrete IL2, IFNγ and TNFβ, whereas TH2 cells, but not TH1 cells, express IL-4, IL-5, IL-6 and IL-10. The molecular mechanisms underlying the evolution of these two different cell types from common precursors are still not completely known. Studies with transgenic mice carrying null mutations of the IL-4 gene have shown that IL-4 plays an important role in the establishment of a functional TH2 immune response. The different patterns of cytokine secretion correspond with different functions as immune effectors. TH1 cells promote cell-mediated effector responses. TH2 cells are mainly helper cells that influence B-cell development and augment humoral responses such as the secretion of antibodies, predominantly of IgE, by B-cells. Both types of TH cells influence each other by the Cytokines they secrete; IFNγ, for example, can downregulate TH2 clones while TH2 cytokines, such as IL-10 can suppress TH1 functions. IFNγ has been shown also to inhibit the proliferation of murine TH2 cells but not that of TH1 helper T-lymphocyte clones. It thus appears that these functional subsets are mutually antagonistic such that the decision of which subset predominates within an infection may determine also its outcome. BMS810FFSA human Th1/Th2 11plex 5 3 PRINCIPLES OF THE TEST 3.1 Principles of the Fluorescent Bead Immunoassay Microspheres are coated with antibodies specifically reacting with each of the analytes to be detected in the multiplex system. The beads can be differentiated by their sizes and by their distinct spectral addresses. Fig. 1 Bead with capture antibody A mixture of coated beads for each analyte to be measured is incubated with the samples or standard mixture. The analytes present in the sample bind to the antibodies linked to the fluorescent beads. Fig. 2 A biotin-conjugated second antibody mixture is added, the specific antibodies bind to the analytes captured by the first antibodies. Fig. 3 - Biotinylated Antibody Streptavidin - Phycoerythrin is added, binds to the biotin conjugate and emits fluorescent signals. Fig. 4 - Streptavidin-PE BMS810FFSA human Th1/Th2 11plex 6 3.2 Principles of the FlowCytomix 1023 Fig. 5 SSC-H Two sets of microspheres of different size are used for the FlowCytomix: Size A: 5.5 µm Size B: 4.4 µm R1 0 R2 0 Size A set consists of 11 bead populations, size B set consists of 9 bead populations internally dyed with different intensities of a fluorescent dye (see Fig. 6 and Fig. 7). Fig. 6 The dye excites with an Argon, He-Ne or even UV laser, and emits in the far red (690 nm) The two different bead sizes make it possible to distinguish 20 bead sets in one fluorescence channel. So 20 different bead sets distinguished by internal dye intensity and bead size allow the simultaneous quantification of 20 analytes in a single small volume sample using the same principle as an ELISA (refer to 3.1). BMS810FFSA human Th1/Th2 11plex Fig. 7 FSC-H 1023 7 4 REAGENTS PROVIDED 1 vial Setup Beads (SB) 11 vials (50 µl) Fluorescent Beads (20x) coated with specific antibodies Table 1 Antibody Specificity Bead Population Clonality h IFN-γ A4 monoclonal h IL-1β B8 monoclonal h IL-2 A6 monoclonal h IL-4 B6 monoclonal h IL-5 B7 monoclonal h IL-6 A12 monoclonal h IL-8 A10 polyclonal h IL-10 A8 monoclonal h IL-12 p70 A2 monoclonal h TNF-α B9 monoclonal h TNF-β B10 monoclonal BMS810FFSA human Th1/Th2 11plex 8 11 vials Standard (lyophilized) Table 2 Standard Concentration upon reconstitution h IFN-γ 400 ng/ml h IL-1β 400 ng/ml h IL-2 400 ng/ml h IL-4 400 ng/ml h IL-5 400 ng/ml h IL-6 400 ng/ml h IL-8 200 ng/ml h IL-10 400 ng/ml h IL-12 p70 400 ng/ml h TNF-α 400 ng/ml h TNF-β 400 ng/ml BMS810FFSA human Th1/Th2 11plex 9 11 vials (100 µl) Biotin-Conjugate (20x) (specific antibody conjugated to biotin) Table 3 Biotin-Conjugate Specificity Clonality h IFN-γ monoclonal h IL-1β monoclonal h IL-2 monoclonal h IL-4 monoclonal h IL-5 monoclonal h IL-6 monoclonal h IL-8 polyclonal h IL-10 monoclonal h IL-12 p70 monoclonal h TNF-α monoclonal h TNF-β monoclonal 1 bottle (50 ml) Assay Buffer (10x) (PBS with 10% BSA) 1 bottle (13 ml) Reagent Dilution Buffer (RDB), ready to use 1 vial (200 µl) Streptavidin-Phycoerythrin BMS810FFSA human Th1/Th2 11plex 10 5 STORAGE INSTRUCTIONS Store kit and components at 2 to 8°C. The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling. 6 SPECIMEN COLLECTION Cell culture supernatant, serum, plasma (heparinized, citrate) and whole blood were tested with this assay. Other biological samples might be suitable for use in the assay. Remove serum or plasma from the clot or cells, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly lipemic specimens. Fat causes agglutination of the beads. Centrifugation of lipemic samples (about 16.000 x g for 5 min) before analysis is recommended. Pay attention to a possible “Hook Effect” due to high sample concentrations (see 15.4.). Clinical samples should be kept at 2° to 8°C and separated rapidly before storing at -20°C to avoid loss of bioactivity. If samples are to be run within 24 hours, they may be stored at 2° to 8°C. Avoid repeated freeze-thaw cycles. BMS810FFSA human Th1/Th2 11plex 11 7 MATERIALS REQUIRED BUT NOT PROVIDED − A flow cytometer equipped with one laser (488 nm or 532 nm) capable of detecting and distinguishing fluorescence emissions at 575 nm and far red (685 - 690 nm) − FlowCytomix Pro Software: The FlowCytomix Pro Software is complimentary and can be ordered at [email protected] (Cat. No. BMS8400FF) or downloaded at www.bendermedsystems.com/software-download. − In the case a filter plate is used, a Filtration manifold is required (NOTE: The filter plate and adhesive films are not included in the sample kit, but in regular FlowCytomix Multiplex kits): We recommend to use the “Multi-Well Plate Vacuum Manifold” (PALL, cat # P/N 5017) for bead washing. The Filtration manifold can be ordered via Bender MedSystems in combination with the FlowCytomix Kit: Multi-Well Plate Vacuum Manifold, PALL (BMS497FF) − Centrifuge − Sample acquisition tubes for a flow cytometer − Aluminium foil − 5 ml and 10 ml graduated pipettes − 10 µl to 1,000 µl adjustable single channel micropipettes with disposable tips − 20 µl to 300 µl adjustable multichannel micropipettes with disposable tips − Multichannel micropipette reservoir − Beakers, flasks, cylinders necessary for preparation of reagents − Glass-distilled or deionized water − Vortex mixer BMS810FFSA human Th1/Th2 11plex 12 8 PRECAUTIONS FOR USE − All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice (www.bendermedsystems.com). − Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. − Do not mix or substitute reagents with those from other lots or other sources. − Do not use kit reagents beyond expiration date on label. − Do not expose kit reagents to light during storage or incubation; the beads and Streptavidin-Phycoerythrin are photosensitive! − Do not pipette by mouth. − Do not eat or smoke in areas where kit reagents or samples are handled. − Avoid contact of skin or mucous membranes with kit reagents or specimens. − Rubber or disposable latex gloves should be worn while handling kit reagents or specimens. − Avoid splashing or generation of aerosols. − In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes. − Use clean, dedicated reagent trays for dispensing the conjugates. − Exposure to acids will inactivate the conjugate. BMS810FFSA human Th1/Th2 11plex 13 − Glass-distilled water or deionized water must be used for reagent preparation. − Decontaminate and dispose of specimens and all potentially contaminated materials as if they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C. − Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0 % sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite. BMS810FFSA human Th1/Th2 11plex 14 9 PREPARATION OF REAGENTS AND SAMPLES All samples except whole blood can be used without predilution. Whole blood samples require at least a 5-fold predilution in Assay Buffer (1x). Bring all reagents to room temperature and vortex well before usage! Calculation of reagent volumes using FlowCytomix Pro Software Before starting the hands-on work this function facilitates the calculation of reagent volumes specifically for the number of samples you choose. Go to “Start/Programs/Bender MedSystems/Test-Setup” to open the calculator. Enter the number of parameters and the number of your samples. Choose single or dual evaluation for both standards and samples. The program automatically provides you with the customized volumes. Please note that the volumes given by the program are the exact volumes required in your assay. We therefore recommend to generously round up the final volumes in order to prepare enough of all reagents and mixtures. 9.1 Assay Buffer Mix the contents of the bottle well. Add contents (50.0 ml) Assay Buffer Concentrate (10x) to 450 ml distilled or deionized water and mix gently to avoid foaming. Store at 2° to 8°C. BMS810FFSA human Th1/Th2 11plex 15 9.2 Preparation of Standard Preparation of Standard Mixture: Determine the number of analytes. It is recommended to centrifuge vials for a few seconds in a microcentrifuge before opening to collect lyophilized standard at the bottom. The lyophilized standard must be reconstituted by adding distilled water according to the label on the standard vial. Swirl vial thoroughly to ensure quantitative solubilization of contents. Wait ten minutes before pipetting the standard. It is recommended to centrifuge vials for a few seconds in a microcentrifuge before pipetting reconstituted standard. Add 10 µl of each reconstituted standard to a vial labelled Standard 1. Fill up to the final volume of 200 µl with Assay Buffer (1x). (This is a 1:20 dilution of each reconstituted standard.) Serial Dilution of Standard Mixture: Add 100 µl Assay Buffer (1x) to 6 tubes labelled standard 2 to 7. Transfer 50 µl of standard 1 to tube 2, mix the contents of tube 2 and transfer 50 µl to tube 3. Repeat the procedure creating a row of 7 standard dilutions. Discard immediately any solubilized or prediluted standard left after usage. Fig. 8 BMS810FFSA human Th1/Th2 11plex 16 9.3 Preparation of Bead Mixture Prepare the Bead Mixture in a centrifuge tube according to the following calculation. a. Per test ( = well) 25 µl of the Bead Mixture is required. Consider tests for standard curves, blanks and samples plus an additional test for standard 1 for setup. Calculate the final volume (V fin) of the Bead Mixture needed. V fin = number of tests x 25 µl Round up for pipetting reservoir. See also Table 4. b. Vortex individual bead vial for 5 seconds and pipette 1/20 of final volume (V fin) of each bead set to a vial labelled “Bead Mix”. e.g.: V fin = 800 µl; 1/20 of final volume = 40 µl c. Fill up to the final volume (V fin) with Reagent Dilution Buffer (RDB). d. Centrifuge at 3000 x g for 5 min. e. Carefully remove excess liquid from the surface, leaving 50 µl in the vial (e.g.: total volume 800 µl, take off 750 µl). Avoid resuspension of beads. f. Add the same volume of Reagent Dilution Buffer that has been removed (e.g.: 750 µl) and vortex for 5 seconds. Table 4 Number of tests 26 Volume of each Bead Set (µl) Volume of RDB (µl) Final volume of Bead Mixture (µl) 40 360 800 BMS810FFSA human Th1/Th2 11plex 17 9.4 Preparation of Biotin-Conjugate Prepare Biotin-Conjugate-Mixture according to the following calculation. a. Per test ( = well) 50 µl of the Biotin-Conjugate-Mixture is required. Consider tests for standard curves, blanks and samples plus an additional test for standard 1 for setup. Calculate the final volume (V fin) of the Biotin-Conjugate-Mixture needed. V fin = number of tests x 50 µl Round up for pipetting reservoir. See also Table 5 b. Pipette 1/20 of final volume (V fin) of each Biotin-Conjugate to a vial labelled “Biotin-Conjugate Mix”. e.g.: V fin = 1600 µl, 1/20 of final volume = 80 µl c. Fill up to the final volume (V fin) with Reagent Dilution Buffer (RDB). Table 5 Number of tests 26 Volume of each Volume of Biotin-Conjugate (µl) RDB (µl) 80 720 Final volume of Biotin-Conjugate Mixture (µl) 1600 9.5 Preparation of Streptavidin-PE Solution Make a dilution of the concentrated Streptavidin-PE solution in Assay Buffer (1x) according to Table 6: Table 6 Number of tests 26 conc. Streptavidin-PE (µl) 64 Assay Buffer (1x) (µl) 1936 Final volume of Streptavidin-PE solution (µl) 2000 The volumes can be adjusted to the number of tests needed. BMS810FFSA human Th1/Th2 11plex 18 10 TECHNICAL TIPPS Please read carefully before you start: 10.1 Mixing When preparing the Bead Mixture, pipette the bead solution all the way down to the bottom of the tube, so that you do not lose any material on the walls of the tube. Inadequate mixing can lead to little or no beads being detected. Vortex the beads immediately before mixing with the standards or samples and again before analysis on the flow cytometer. Vortex each sample tube for 3-5 seconds before placing the tube on the flow cytometer as this will yield better discrimination of the bead populations in the FL3/FL4 channel. 10.2 To avoid double bead populations in the analysis plot When applying the bead solution to the assay plate, put the beads into the standard- / sample solution in the well to make sure that the beads do not stick on the side of the tube/well. (Beads which do not come in contact with the detection antibody during the incubation step will show a much lower PE signal. These beads will lead to a second, almost “PE-negative” population.) 10.3 To optimize sensitivity It is critical that all steps after the addition of the detector reagents are protected from light to obtain maximum fluorescence of the PE reporter system. When establishing the instrument settings with Standard one (highest concentration), it is essential to place the bead populations at the very right margin of the acquisition plot. Thereby the distribution of all standard concentrations across all four decades in FL-2 will be optimized. 10.4 Blocked filter plate Serum or plasma samples can sometimes block the filter plate during washing. If this occurs very carefully push a needle through the hole underneath the respective well of the plate. Do not stick the needle all the way through the filter as this will damage the membrane! BMS810FFSA human Th1/Th2 11plex 19 11 TEST PROTOCOL 11.1 Test Procedure Using the Filter Plate NOTE: The filter plate and adhesive films are not included in the sample kit, but in regular FlowCytomix Multiplex kits. In this case a filtration manifold (see chapter 7) is required for test performance. For the FlowCytomix sample kit using tubes please refer to chapter 11.2. Prepare Assay Buffer referring to 9.1. Prepare Biotin-Conjugate, Bead Mixtures and standards referring to 9.4, 9.3 and 9.2. a. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards and calculate amount of reagents needed respectively (see chapter 9). Place adhesive film over unused wells so that vacuum filtration works efficiently. We highly recommend to prepare 2 standard curves and an additional standard 1 (see Table 7Fehler! Verweisquelle konnte nicht gefunden werden. for pipetting scheme) in order to have enough solution of standard 1 also for cytometer setup (section 12 of this manual). b. Add 50 µl Assay Buffer (1x) to the filter plate to pre-wet the wells. Aspirate using the vacuum filtration manifold. Blot the bottom of the plate after filtration. c. Add 25 µl of Standard Mixture dilutions 1 to 7 to designated wells of the plate (see Table 7). BMS810FFSA human Th1/Th2 11plex 20 Table 7 An example of the arrangement of blanks, standards and samples in the microwell strips: 1 2 3 A Standard 1 Standard 1 Standard 1 for Setup B Standard 2 Standard 2 Sample 1 C Standard 3 Standard 3 Sample 2 D Standard 4 Standard 4 Sample 3 E Standard 5 Standard 5 ……….. F Standard 6 Standard 6 ……….. G Standard 7 Standard 7 ……….. H Blank Blank ……….. d. Add 25 µl of Assay Buffer (1x) to the blank wells. e. Add 25 µl of Standard Mixture dilution 1 to well A3 (see Table 7) which is designated for cytometer setup (highly recommended). f. Add 25 µl of sample to the designated wells. g. Add 25 µl of Bead Mixture (refer to Preparation of Bead Mixture 9.3) to all wells, including the blank wells. h. Add 50 µl of Biotin-Conjugate Mixture (refer to Preparation of Biotin-Conjugate 9.4) to all wells, including the blank wells. i. Cover wells with adhesive film, avoid putting an excessive pressure on the top of the plate. Protect from light with an aluminium foil and incubate at room temperature (18° to 25°C) for 2 hours on a microplate shaker at 500 rpm. j. Prepare Streptavidin-PE Solution (refer to Preparation of Streptavidin-PE 9.5). BMS810FFSA human Th1/Th2 11plex 21 k. Remove adhesive film and empty wells using the vacuum filtration manifold. Add 100 µl Assay Buffer (1x) to microwell strips and empty wells again using the vacuum filtration manifold. Repeat this step once. Remove any liquid on the bottom of the filter plate with an absorbent towel. Remove towel before next step. l. Add 100 µl Assay Buffer (1x) to each well. m. Add 50 µl of Streptavidin-PE Solution to all wells including the blank wells. n. Cover with an adhesive film, avoid putting an excessive pressure on the top of the plate. Protect from light with an aluminium foil and incubate at room temperature (18° to 25°C) for 1 hour on a microplate shaker at 500 rpm. o. Remove adhesive film and empty wells using the vacuum filtration manifold. Add 100 µl Assay Buffer (1x) to microwell strips and empty wells again using the vacuum filtration manifold. Repeat this step once. Remove any liquid on the bottom of the filter plate with an absorbent towel. Remove towel before next step. p. Add 200 µl Assay Buffer (1x) to each well. q. Mix the contents of each well by repeated aspiration and ejection, and transfer these 200 µl of each well into a separate sample acquisition tube for a flow cytometer and fill up to 500 µl final volume by adding 300 µl of Assay Buffer (1x). r. Proceed to next step of protocol or store tubes for a maximum of 24 hours at 2 – 8°C protected from light. Swirl tube contents thoroughly if they are stored longer than 2 hours. s. Before analysing samples on a flow cytometer make cytometer setup (refer to chapter 12) BMS810FFSA human Th1/Th2 11plex 22 11.2 Test Procedure Using Tubes In this case incubations are performed in tubes. Centrifugation steps are necessary for test performance. Prepare Assay Buffer referring to 9.1. Prepare Biotin-Conjugate, Bead Mixtures and standards referring to 9.4, 9.3 and 9.2. a. Determine the number of cytometer tubes required to test the desired number of samples plus appropriate number of tubes needed for running blanks and standards and calculate amount of reagents needed respectively (see chapter 9). We highly recommend to prepare 2 standard curves and an additional standard 1 (see Table 7 for pipetting scheme) in order to have enough solution of standard 1 also for cytometer setup (section 12 of this manual). b. Add 25 µl of Standard Mixture dilutions 1 to 7 in designated tubes (see Table 7). c. Add 25 µl of Assay Buffer (1x) to the blank tubes. d. Add 25 µl of Standard Mixture dilution 1 to tube 17 which is designated for cytometer setup (highly recommended). e. Add 25 µl of sample to the designated sample tubes. f. Add 25 µl of Bead Mixture (refer to Preparation of Bead Mixture 9.3) to all tubes, including the blank tubes. g. Add 50 µl of Biotin-Conjugate Mixture (refer to Preparation of Biotin-conjugate 9.4) to all tubes, including the blank tubes. h. Mix the contents of each tube well and incubate at room temperature (18° to 25°C) for 2 hours. Protect from light with an aluminium foil. i. Prepare Streptavidin-PE Solution (refer to Preparation of Streptavidin-PE 9.5). j. Add 1 ml of Assay Buffer (1x) to all tubes and spin down at 200 x g for 5 minutes. BMS810FFSA human Th1/Th2 11plex 23 k. Carefully discard the supernatant from each tube leaving 100 µl of liquid in each tube. l. Repeat steps j. and k. m. Add 50 µl of Streptavidin-PE to all tubes including the blank tubes. n. Mix the contents of each tube well and incubate at room temperature (18° to 25°C) for 1 hour. Protect from light with an aluminium foil. o. Add 1 ml of Assay Buffer (1x) to all tubes and spin down at 200 x g for 5 minutes. p. Carefully discard the supernatant from each tube leaving 100 µl of liquid in each tube. q. Repeat steps o and p. r. Add 500 µl Assay Buffer (1x) to each tube. s. Proceed to next step of protocol or store tubes for a maximum of 24 h at 2 – 8°C protected from light. Swirl tube contents thoroughly if they are stored longer than 2 hours. t. Before analysing samples on a flow cytometer make cytometer setup (refer to chapter 12). BMS810FFSA human Th1/Th2 11plex 24 12 CYTOMETER SETUP 12.1 Preparation of Cytometer Setup Beads Vortex the vial with setup beads for several seconds. Pipette 500 µl of setup beads into a tube labelled SB. 12.2 Instrument Setup Please note before setup: The setup beads provided with this assay are needed • to adjust FS/SSC parameters • to create regions for the bead populations • to define the number of events counted • to adjust voltage of FL-2 so that the bead populations are positioned in the left part of the dot blot. This ensures that the bead populations of Standard 1 will be visible on the screen. With every new experiment before starting sample acquisition, adjust voltage of FL-2/FL-3(-4) and compensation with standard 1 in “Setup Mode” (prepared twice for this purpose, see 10), which gives the highest PE (FL-2) signal. After all settings and compensation have been optimized, switch to “Acquisition Mode” and start measuring standards and samples. • Do not change Voltage and Compensation, nor Flow Through (small – medium – high) during measurement. For the most commonly used flow cytometers (e.g. Becton Dickinson or DakoCytomation) you can immediately start cytometer setup as below. If you run a FlowCytomix assay on a FC500 Instrument from Beckman Coulter you must ensure that the Forward Scatter (FS) measurements are collected at 1-8°. To accomplish this you must insert the FS 1-8° Field Stop into place: remove the front cover to locate the FS 1-8° Field Stop. Slide the knob from the right (= default 1-19° position) to the left and push to lock in place. On the XL Series systems the EPICS XL/XL-MCL FS Low Angle Collection Kit is recommended to accomplish this. Then proceed to general protocol. BMS810FFSA human Th1/Th2 11plex 25 a. Perform instrument start up following the manufacturer’s recommendations. b. Perform flow check to verify alignment and fluidic stability of the system. c. Open a new protocol. d. Create a Dot Plot window with FS (Forward Scatter) for X- and SSC (Side Scatter) for Y-axis. e. Set FS and SSC to linear mode. f. Create a second and a third Dot Plot window with FL-2 for X- and FL3 (-4) for Y-axis. NOTE: The FL channel number for the specific nm range depends on the instrument. e.g.: 690 nm is FL-3 in a BD FACSCalibur but FL- 4 in a Beckman Coulter Flow Cytometer g. Set FL-2 and FL-3 (-4) to Log mode. h. Set Compensation to zero. i. Save your protocol frequently during setup. j. In “Setup Mode” run tube SB (with setup beads). k. Adjust the parameters of FS and SSC so that both bead populations in the control are visible in the opened window (see Fig. 9). Fig. 9 (Pause and restart acquisition frequently during the cytometer setup in order to reset detected values after setting adjustment.) BMS810FFSA human Th1/Th2 11plex 26 l. Create regions “R1” for the large beads and “R2” for the small beads (see Fig. 10). Fig. 10 Define number of events so that 300 events per analyte are measured within gate R2 (small beads) e.g. Count 1500 events of gated bead populations for the 10-plex (300 events per analyte – 5 small bead populations 1500 events). BMS810FFSA human Th1/Th2 11plex 27 m. Select gate R1 in the first FL-2/FL-3(-4) Dot Plot (Fig. 11, Gate R1). Select gate R2 in the second FL-2/FL-3(-4) Dot Plot (see Fig. 11, Gate R2). Fig. 11 Gate R1 Gate R2 Adjust the parameters of FL-2 and FL-3 (-4) so that the bead populations are visible in the opened window. With setup beads of tube SB voltage of FL-2 has to be adjusted so that the bead populations are positioned in the left part of the dot blot. This ensures that the bead populations of standard 1 will be visible on the screen. n. Save your protocol frequently during setup. BMS810FFSA human Th1/Th2 11plex 28 o. Before starting the acquisition of standards and samples stay in SETUP MODE and adjust the settings using standard 1, which gives the highest PE (FL-2) signal. Adjust the parameters of FL-2 so that the bead population with the highest PE (FL-2) signal sticks to the right axis while the whole population is visible (see Fig. 12). Fig. 12 Gate R1 Gate R2 IL-6 IL-8 IL-10 IL-2 IFN-g IL-12 p70 TNF-b TNF-a IL-5 IL-4 IL-1b p. Restart acquisition of standard 1 for setup. (Pause and restart acquisition frequently during the cytometer setup in order to reset detected values after setting adjustment) BMS810FFSA human Th1/Th2 11plex 29 q. If the bead populations are not in a horizontal position (as shown in Fig. 13) increase compensation setting for FL-3 (-4) - %FL-2. Fig. 13 Gate R1 Gate R2 FL-3 (-4) - %FL-2 BMS810FFSA human Th1/Th2 11plex 30 r. If the bead populations are not in a horizontal position (see Fig. 14) decrease compensation setting for FL-3 (-4) - %FL-2. Fig. 14 Gate R1 GateR2 FL-3 (-4) - %FL-2 A final setup may look like that shown in section 12.3 of this manual. s. Save your protocol. BMS810FFSA human Th1/Th2 11plex 31 12.3 Acquisition a. Switch from SETUP MODE to ACQUISITION MODE. b. To simplify auto file loading in the analysis software save all measured data with the same file name and consecutive numbering (e.g.: FBI.001, FBI.002, FBI.003, …). c. Begin analysing samples starting with the standard curve (S1-S7 and blank), followed by the samples. A final setup (e.g.: for BD FACSCalibur) may look like that shown in Table 8: N.B.: individual setup is required Table 8 Detector FS SSC FL1 FL2 FL3 FL1 FL2 FL2 FL3 Voltage E00 350 600 650 610 Compensation 0.0 0.0 0.0 7.5 Amp 5.4 2 1 - FL2 FL1 FL3 FL2 BMS810FFSA human Th1/Th2 11plex Mode Lin Lin Lin Log Log 32 A final setup (e.g.: for BD FACScan) may look like that shown in Table 9: N.B.: individual setup is required Table 9 Detector FS SSC FL1 FL2 FL3 FL1 FL2 FL2 FL3 Voltage E01 336 163 316 501 Compensation 0.0 0.0 0.0 10.0 Amp 1.9 1 - Mode Lin Lin Log Log Log FL2 FL1 FL3 FL2 A final setup (e.g.: for BC FC500) may look like that shown in Table 10: N.B.: individual setup is required Table 10 FS SSC FL1 FL2 FL3 FL4 FL5 Detectors 200-400 200-400 200-400 500 250 580 250 Compensation FL1 FL1 FL2 FL3 FL4 FL5 BMS810FFSA human Th1/Th2 11plex Gain 20 5 to 10 1 1 1 1 1 FL2 10 33 13 CALCULATION OF RESULTS For calculation of results refer to the BMS FlowCytomix Software manual. BMS FlowCytomix Pro software and manual is included in the kit, but also available for free download on: www.bendermedsystems.com Table 11 Representative standard curves. Do not use these curves to derive test results. A standard curve must be run for each group of samples assayed. Conc. Fluorescent Intensity (FI) pg/ml h IL-1β h IL-2 h IL-4 h IL-5 h IL-6 h IL-10 h IL-12 p70 20000 273.0 288.2 94.9 254.0 501.0 255.1 246.1 6667 112.1 143.0 28.7 96.0 374.2 126.5 128.8 2222 38.6 56.1 8.0 32.5 182.5 53.5 53.6 741 12.4 15.1 3.0 11.8 80.8 21.3 21.3 247 4.9 4.2 1.2 4.5 33.9 9.4 8.4 82 2.8 1.6 0.74 2.2 16.9 4.8 3.8 27 1.6 1.0 0.66 1.3 9.8 3.0 2.1 0 0.9 0.7 0.6 0.6 4.3 1.7 0.6 Conc. Fluorescent Intensity (FI) Conc. FI pg/ml h IFN-γ h TNF-α h TNF-β pg/ml h IL-8 20000 220.0 205.6 464.3 10000 384.2 6667 93.9 98.2 362.7 3333 263.5 2222 37.9 42.9 214.7 1111 119.3 741 15.2 17.6 104.3 370 47.6 247 6.3 7.5 44.4 123 20.0 82 3.0 3.8 20.2 41 9.6 27 1.9 2.3 12.0 14 6.1 0 0.8 1.2 4.4 0 3.3 BMS810FFSA human Th1/Th2 11plex 34 − To determine the concentration of circulating analytes for each sample, first find the fluorescent intensity value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding concentration. − It is suggested that each testing facility establishes a control sample of known concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid. BMS810FFSA human Th1/Th2 11plex 35 14 LIMITATIONS − Since exact conditions may vary from assay to assay, standard curves must be established for every run. − Bacterial or fungal contamination of either samples or reagents or cross-contamination between reagents may cause erroneous results. − Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. BMS810FFSA human Th1/Th2 11plex 36 15 PERFORMANCE CHARACTERISTICS 15.1 Sensitivity The limit of detection of each analyte defined as the analyte concentration resulting in a fluorescent intensity significantly higher than that of the dilution medium (mean + 2 standard deviations) was determined to be: Table 12 Analyte Sensitivity (pg/ml) h IFN-γ 1.6 h IL-1ß 4.2 h IL-2 16.4 h IL-4 20.8 h IL-5 1.6 h IL-6 1.2 h IL-8 0.5 h IL-10 1.9 h IL12p70 1.5 h TNF-α 3.2 h TNF-ß 2.4 BMS810FFSA human Th1/Th2 11plex 37 15.2 Reproducibility 15.2.1 Intra-assay Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 4 serum samples containing different concentrations (high, medium high, medium low and low concentration) of all analytes. Two standard curves were run with each assay. Data below show the mean intra-assay coefficient of variation for each analyte (see Table 13). Individual user data may vary due to differences in protein content of serum/plasma pools or individual donor plasma/serum. Table 13 The mean intra-assay coefficient of variation for each analyte Analyte CV Sample 1 high (%) CV Sample 2 medium high (%) CV Sample 3 medium low (%) CV Sample 4 low (%) Mean intraassay CV (%) h IFN-γγ 4.5 3.5 3.8 12.8 6.2 h IL-1β β 4.3 4.9 6.9 9.6 6.4 h IL-2 6.5 5.5 10.5 14.1 9.2 h IL-4 9.0 8.4 6.0 7.0 7.6 h IL-5 4.0 5.8 7.5 7.8 6.3 H IL-6 6.9 7.9 12.4 10.1 9.3 h IL-8 6.6 3.0 7.8 16.4 8.5 h IL-10 7.4 10.7 20.1 21.5 14.9 h IL-12 p70 4.8 2.6 4.3 6.5 4.5 h TNF-α α 8.0 9.7 12.4 18.0 12.0 h TNF-β β 3.1 2.6 4.0 16.0 6.4 BMS810FFSA human Th1/Th2 11plex 38 15.2.2 Inter-assay Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 4 serum samples containing different concentrations of all analytes (high, medium high, medium low and low concentration). Two standard curves were run with each assay. Data below show the mean interassay coefficient of variation for each analyte (see Table 14). Individual user data may vary due to differences in protein content of serum/plasma pools or individual donor plasma/serum. Table 14 The mean inter-assay coefficient of variation for each analyte Analyte CV Sample 1 high (%) CV Sample 2 medium high (%) CV Sample 3 medium low (%) CV Sample 4 low (%) Mean interassay CV (%) h IFN-γγ 2.5 3.7 4.6 17.4 7.1 h IL-1β β 4.2 5.2 9.4 4.0 5.7 h IL-2 4.3 0.5 1.9 10.5 4.3 h IL-4 6.7 2.8 2.3 9.7 5.4 h IL-5 5.8 2.1 2.5 2.8 3.3 H IL-6 1.9 2.2 6.6 12.3 5.7 h IL-8 1.8 2.5 4.0 17.3 6.4 h IL-10 10.2 7.1 17.1 31.0 16.3 h IL-12 p70 1.7 1.7 6.2 4.0 3.4 h TNF-α α 8.3 6.4 7.4 13.3 8.9 h TNF-β β 0.9 2.7 10.3 8.9 5.7 BMS810FFSA human Th1/Th2 11plex 39 15.3 Specificity There was no detectable crossreactivity observed for other combinable analytes of Simplex and Multiplex Assays from Bender MedSystems. (For detailed information refer to “Combination Table“ on www.bendermedsystems.com.) 15.4 Hook effect Samples with expected concentrations two fold higher than the standard of the highest concentration should be diluted 10 fold in Assay Buffer (1x) before assay performance to prevent false negative results due to “hook effects”. BMS810FFSA human Th1/Th2 11plex 40 16 FAQ FOR FLOWCYTOMIX KITS Is there a particular flow cytometer required for the use of FlowCytomix Kits? The FlowCytomix microspheres are detectable in the most commonly used flow cytometers. The flow cytometer needs to be equipped with one laser (488 nm or 532 nm) capable of detecting and distinguishing fluorescence emissions at 575 nm and far red (685 nm - 690 nm). FlowCytomix suitability has been tested for: Beckman Coulter: EPICS® XLTM / XL-MCL TM Cytomics TM FC500 (see section 12.2 of this booklet) (to resolve the two bead populations, the Forward Scatter collection angles have to be at the correct position. Move the Field Stop mechanism to the left position in order to change the Forward Scatter collection angles from 1-19 degrees to 1-8 degrees.) BD FACScan TM BD FACSCalibur TM BD FACSCanto TM BD TM LSR I BD TM LSR II Is there a special software required? Yes, the Flow raw data files (FCS, LMD) are analysed with the specifically designed FlowCytomixPro Software. The software is included in the FlowCytomix Multiplex Kits and the FlowCytomix Basic Kits and can be downloaded from the web page: www.bendermedsystems.com Does the software interfere with the flow cytometer operating system? Software has been virus checked with latest version of McAfee VirusScan. FlowCytomixPro Software has been successfully operated in conjunction with all commonly used flow cytometer models. It does not interfere with the flow cytometer operating system. BMS810FFSA human Th1/Th2 11plex 41 In addition the FlowCytomixPro Software does not need to be installed on the flow cytometer working station; data files can be transferred to any other computer. What happens if less than 11 analytes have to be measured? Simplex Kits are available for this purpose. Simplex Kits are designed for the detection of one specific analyte. Simplex Kits can be combined to give any combination (within the human or mouse range) and number of analytes to be measured. In which case is a Basic kit required? A Basic Kit is required for any combination of Simplex Kits. In order to run a FlowCytomix assay some reagents are needed only once, even in the case of combining several Simplex Kits. These reagents are provided in a Basic Kit. Are the assays performed in plates or in tubes? The FlowCytomix assays can be either performed like an ELISA in a 96well filter plate, in this case a filtration manifold is required, or alternatively in tubes, which requires centrifugation of the tubes during the washing procedure. Why is a cytometer setup required before measuring standards and samples? The cytometer setup is required by the mode of operation of the flow cytometer. Setup beads are included in the Basic Kits and in the Multiplex Kits. With every new experiment, before starting the acquisition of standards and samples stay in SET UP MODE and adjust the settings using the highest Standard 1. What wavelength is chosen for measurement? The maximum emission of PE is at 578 nm and the emission of the beads is in the far red region (685 - 690 nm). Is it possible to measure with high “flow through” settings? There are three flow through settings: low-medium-high. Increasing the “flow through” may result in a dispersed bead population. Therefore it is recommended to start with the low “flow through”; it can only be increased if the bead population does not start to disperse. BMS810FFSA human Th1/Th2 11plex 42 Once the final instrument settings are saved, do not change the “flow through” during measurement. Does a FlowCytomix Kit work on a LuminexTM instrument? No, the FlowCytomix Kit can only be measured on a flow cytometer. Is it correct to use the non linear part of the standard curve? Yes, it is possible to use the non linear part of the standard curve for calculation of results. Dilutions of samples behave in the same way as the standard curve. FAQ´s regarding Combination: How many analytes can be combined in one assay? Up to 20 analytes are possible. Which analytes can be combined? There are 20 different bead sets that can be distinguished in the FlowCytomix assay. Analytes containing different bead sets can be analysed simultaneously. You can find the analytes and the corresponding bead set listed at “4 Reagents Provided”. Can Multiplex kits be combined with each other? Multiplex Kits can be combined with each other as long as they do not use the same bead sets. Pay attention to the kind of Conjugate (PE or Biotin). For detailed information about possible combinations refer to “Combination Table“ on www.bendermedsystems.com. Can Simplex kits be combined with a Multiplex kit? It is possible in case all analytes contain different bead sets. Pay attention to the kind of Conjugate (PE or Biotin). For detailed information about possible combinations refer to “Combination Table“ on www.bendermedsystems.com. Can components from different lots be mixed? The bead sets, standards and conjugates are lot-specific and must be used in combination with each other. Do not mix these components from different kit lots. Assay Buffer, Reagent Dilution Buffer and Streptavidin-PE are not lotspecific and can therefore be exchanged between different kit lots. BMS810FFSA human Th1/Th2 11plex 43 Is it possible to combine Biotin Conjugates with PE-conjugates in one conjugate mixture? In case you are using at least one Biotin-Conjugate in your combination a further incubation step with Streptavidin-PE is necessary. This does not interfere with the PE-conjugates. BMS810FFSA human Th1/Th2 11plex 44 17 BIBLIOGRAPHY 1) Bloom BR et al. Revisiting and revising T suppressor cells. Immunology Today 13: 131-6 (1992). 2) Bottomly K. A functional dichotomy in CD4 + T lymphocytes. Immunology Today 9: 268-74 (1988). 3) Cherwinski HM et al. Two types of mouse helper T cell clone. III. Further differences in lymphokine synthesis between Th1 and Th2 clones revealed by RNA hybridization, functionally monospecific bioassays, and monoclonal antibodies. Journal of Experimental Medicine 166: 1229-44 (1987). 4) Clerici M and Shearer GM A. Th1 to Th2 switch is a critical step in the etiology oh HIV infections. Immunology Today 14: 107-111 (1993). 5) Del Prete GF et al. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. Journal of Clinical Investigation 88: 346-50 (1991). 6) Fiorentino DF et al. IL10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. Journal of Immunology 146: 3444-51 (1991). 7) Firestein GS et al. A new murine CD4 + T cell subset with an unrestricted cytokine profile. Journal of Immunology 143: 518-25 (1989). 8) Gajewski TF and Fitch FW. Anti-proliferative effect of IFN-gamma in immune regulation. I. IFN-gamma inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. Journal of Immunology 140: 4245-52 (1988). 9) Hikida M et al. Suppression of interleukin 4 production from type 2 helper T cell clone by antisense oligodeoxynucleotide. Immunology Letters 34: 297-302 (1992). 10) Kamogawa Y et al. The relationship of IL4 and IFN-gammaproducing T cells studied by lineage ablation of IL4 producing cells. Cell 75: 985-95 (1993). BMS810FFSA human Th1/Th2 11plex 45 11) Kawakami K and Parker DC. Differences between T helper cell lines in signaling pathways for induction of contact-dependent T cell help. European Journal of Immunology 22: 85-93 (1992). 12) Kelso A et al. Heterogeneity in lymphokine profiles of CD4 + and CD8 + T cells and clones activated in vivo and in vitro. Immunological Reviews 123: 85-114 (1991). 13) Lederer JA et al. Regulation of cytokine gene expression in T helper cell subsets. Journal of Immunology 152: 77-86 (1994). 14) Mosmann TR et al. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. Journal of Immunology 136: 2348-57 (1986). 15) Mosmann TR and Coffman RL. Heterogeneity of cytokine secretion patterns and functions of helper T cells. Advances in Immunology 111-47 (1989). 16) Mosmann TR and Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annual Review of Immunology 7: 145-73 (1989). 17) Paul WE and Seder RA. Lymphocyte responses and cytokines. Cell 76: 241-51 (1994). 18) Rocken M et al. A common precursor for CD4 + T cells producing IL2 or IL4. Journal of Immunology 148: 1031-6 (1992). 19) Romagnani S. Type 1 T helper and type 2 T helper cells: functions, regulation and role in protection and disease. International Journal of Clin. Lab. Research 21: 152-8 (1991). 20) Romagnani S. Human TH1 and TH2 subsets: doubt no more. Immunology Today 12: 256-7 (1991). 21) Rooney JW et al. A common factor regulates both Th1- and Th2specific cytokine gene expression. EMBO Journal 13:625-33 (1994). 22) Williams ME et al. Activation of functionally distinct subsets of CD4 + T lymphocytes. Research Immunology 142: 23-8 (1991). For literature update refer to www.bendermedsystems.com BMS810FFSA human Th1/Th2 11plex 46 18 ORDERING INFORMATION For orders please contact: Europe-Headquarters Bender MedSystems GmbH Campus Vienna Biocenter 2 A-1030 Vienna, Austria, Europe phone: +43 1 796 40 40 ext. 114 fax: +43 1 796 40 40 ext. 400 e-mail: [email protected] USA Bender MedSystems, Inc. 849 Hinckley Road Burlingame, CA 94010, USA toll-free phone: +1 (866) 952 2112 toll-free fax: +1 (877) 952 2112 phone: +1 (650) 952 2112 fax: +1 (650) 952 2252 e-mail: [email protected] For technical information please contact: e-mail: [email protected] www.bendermedsystems.com Cat.No. BMS810FFSA human Th1/Th2 11plex BMS810FFSA human Th1/Th2 11plex 47 19 REAGENT PREPARATION SUMMARY 19.1 Assay Buffer Table 15 Assay Buffer Concentrate (10x) (ml) 50 Distilled Water (ml) 450 19.2 Biotin-Conjugate Make a 1:20 dilution of each Biotin-Conjugate creating a Mixture of Conjugates. 19.3 Bead Mixture Make a 1:20 dilution of each Bead Set creating a mixture of beads. Wash Bead Mixture once with Reagent Dilution Buffer 19.4 Standard a. Centrifuge vials for a few seconds to collect lyophilized standard at the bottom. Reconstitute the standard by adding distilled water according to the label on the standard vial. b. Make a 1:20 dilution of the reconstituted standards all diluted in the same vial to get standard 1. Make further serial dilutions. 19.5 Streptavidin-PE Solution Table 16 Number of tests 26 conc. Streptavidin-PE (µl) Assay Buffer (1x) (µl) Final volume of Streptavidin-PE Solution (µl) 64 1936 2000 BMS810FFSA human Th1/Th2 11plex 48 20 TEST PROTOCOL SUMMARY 20.1 USING THE FILTER PLATE: vacuum manifold required − Prepare Assay Buffer − Prepare Biotin-Conjugate Mixture, Bead Mixture and Standard Mixture − Pre-wet microwell strips of filter plate with Assay Buffer (1x) − Pipette 25 µl diluted Standard Mixture dilution 1-7 into designated wells − Add 25 µl Assay Buffer (1x) to the blank wells − Add 25 µl of Standard Mixture dilution 1 to well A3 which is designated for cytometer setup (highly recommended) − Add 25 µl sample to designated wells − Add 25 µl Bead Mixture to all wells − Add 50 µl of Biotin-Conjugate Mixture to all wells − Cover microwell strips, protect from light and incubate 2 hours at room temperature (18° to 25°C) on microplate shaker (500rpm) − Prepare Streptavidin-PE Solution − Wash microwell strips twice with Assay Buffer (1x) using the vacuum filtration manifold − Add 100 µl of Assay Buffer (1x) − Add 50 µl of diluted Streptavidin-PE Solution to all wells − Cover microwell strips, protect from light and incubate 1 hour at room temperature (18° to 25°C) on microplate shaker (500rpm) − Wash microwell strips twice with Assay Buffer (1x) using the vacuum filtration manifold − Add 200 µl of Assay Buffer (1x) BMS810FFSA human Th1/Th2 11plex 49 − Mix the contents of each well by repeated aspiration and ejection, and transfer these 200 µl of each well into a separate sample acquisition tube for a flow cytometer. Fill up to 500 µl final volume with Assay Buffer (1x) − Analyse samples on a flow cytometer. 20.2 USING TUBES: centrifugation steps required − Prepare Assay Buffer − Prepare Biotin-Conjugate Mixture, Bead Mixture and Standard Mixtures − Pipette 25 µl diluted Standard Mixture dilution 1-7 into designated tubes − Add 25 µl Assay Buffer (1x) to the blank tubes − Add 25 µl of Standard Mixture dilution 1 to the tube which is designated for cytometer setup (highly recommended) − Add 25 µl sample to designated tubes − Add 25 µl Bead Mixture to all tubes − Add 50 µl of Biotin-Conjugate Mixture to all tubes − Incubate 2 hours at room temperature (18° to 25°C) protect from light − Prepare Streptavidin-PE Solution − Add 1 ml Assay Buffer (1x) to all tubes − Wash tubes twice by centrifugation (5 min at 200 x g) and discard supernatant carefully leaving 100 µl of liquid in each tube − Add 50 µl of diluted Streptavidin-PE Solution to all tubes − Incubate 1 hour at room temperature (18° to 25°C) protect from light − Add 1 ml Assay Buffer (1x) to all tubes BMS810FFSA human Th1/Th2 11plex 50 − Wash tubes twice by centrifugation (5 min at 200 x g) and discard supernatant carefully leaving 100 µl of liquid in each tube − Add 500 µl Assay Buffer (1x) to all tubes − Analyse samples on a flow cytometer BMS810FFSA human Th1/Th2 11plex