ניסוי: הכרת מכשור מעבדתי

Transcription

‫הטכניון – מכון טכנולוגי לישראל‬
‫הפקולטה להנדסה ביו‪-‬רפואית‬
‫מעבדה בסיסית‪ -‬הכרה של מכשור בביו חומרים‬
‫) ‪(335001‬‬
‫ניסוי‪ :‬הכרת מכשור מעבדתי‬
‫חלק א'‬
‫‪ .1‬מטרות המעבדה‬
‫‪ .2‬תקציר הניסוי‬
‫חלק ב'‬
‫‪ .3‬רקע‪ ,‬מושגים ונספחים‪.‬‬
‫‪ .4‬דו"ח מכין ושאלות הכוונה‪.‬‬
‫חלק ג'‬
‫‪ .5‬תיאור מהלך מערכת הניסוי‬
‫‪ .6‬דרישות הדו"ח המסכם‬
‫ניסוי ‪ :‬ציטומטר זרימה‬
‫עמוד ‪ 1‬מתוך ‪25‬‬
‫עדכון אחרון ‪ :15/02/2015‬ע"י שחר בן שאול‬
‫חלק א'‬
‫‪ .1‬מטרות המעבדה‪:‬‬
‫א‪ .‬הכרת המכשור המעבדתי והשימוש בו‪.‬‬
‫בטיחות‪ :‬עבודה עם חומרים כימים‪ ,‬מנדף ביולוגי אל מול מנדף כימי‪.‬‬
‫‪‬‬
‫שמירה על סטריליות עבודה‪,‬שימוש באתנול ואקונומיקה‪ ,‬סוגי כפפות‪.‬‬
‫‪‬‬
‫שימוש בפיפטורים מכאנים (בגדלים שונים) וחשמליים‪ ,‬שימוש בטיפים‬
‫‪‬‬
‫(גדלים שונים)‪.‬‬
‫הכרת הצנטריפוגות השונות‪ ,‬מהירותן ושימוש במבחנות המתאימות‪.‬‬
‫‪‬‬
‫עבודה עם מקרוסקופ פלורוסנטי‪ ,‬הסתכלות על קווי תאים פלורוסנטים‬
‫‪‬‬
‫(חשיפות שונות וצילום)‪.‬‬
‫עבודה עם ספקטרופוטומטר‪ ,‬עקומת כיול וכימות חלבון בסרום על בסיס‬
‫‪‬‬
‫‪.Bradford protein assay‬‬
‫ב‪ .‬הקניית טכניקות עבודה עם תרביות תאים‪.‬‬
‫‪ ‬הכרות עם סוגי קווי תאים שונים (סרטניים אל מול "נורמאלים")‪.‬‬
‫‪ ‬למידת טכניקות שונות לפיצול וריכוז קווי תאים‪.‬‬
‫‪ ‬כימות תאים חיים על סמך ‪.Trypan blue assay‬‬
‫‪ .2‬תקציר הניסוי‬
‫במעבדה הנ"ל הדגש יהיה על תרגול אישי ומעשי של הסטודנטים‪ .‬במעבדה נעבור במספר תחנות‬
‫ונבצע ניסויים שונים‪ .‬בתחילת המעבדה תערך סקירה על מכשירי המעבדה האנליטיים ואת הציוד‬
‫המעבדתי בהם נשתמש‪ .‬יובהרו היתרונות\חסרונות וכן הרלבנטיות בשימוש בציוד המעבדתי‪.‬‬
‫בהמשך‪ ,‬יוצגו מספר קווי תאים והסבר על מקורם‪,‬צורת גדילתם כ ושימוש בהם במחקר ובתעשייה‪.‬‬
‫בשלב הבא נשתמש בטכניקות שונות להסרת תאים מצלחת גידול (ביולוגי‪-‬טריפסין; מכאנית‪ -‬גירוד‪,‬‬
‫פיטטציה ושאיבה)‪ ,‬סרכוזם בצנטריפוגה ומיהולם ב‪ PBS -‬לנפח עבודה רצוי‪ .‬לשם פיצול התאים‪,‬‬
‫נמדוד את כמות התאים החיים ונמצא את ריכוזם בעזרת ‪ .Trypan blue assay‬בניסוי הבא‪,‬‬
‫נשתמש במיקרוסקופ אור ופלורוסנטי כדי לבחון גודלם וצורתם של קווי תאים השונים‪ .‬הדגש יושם‬
‫על ‪ 2‬קווי תאים פלורוסנטים (אדום‪ RFP-‬וירוק‪ )GFP-‬נחשוף את צמצם המצלמה לזמנים שונים‪,‬‬
‫עמוד ‪ 2‬מתוך ‪25‬‬
‫ בניסוי הבאנשתמש בספקטרופוטומטר לכימות ריכוז החלבון‬.‫נלמד כיצד להחסיר רקע מצילום‬
‫ נמהל מספר מבחנות עם חלבון‬,Bradford ‫ לשם כך נשתמש במבחן‬.‫בסרום בעזרת עקום כיול‬
.FBS -‫ כדי ליצור עקומת כיול וביחס אליה נכמת חלבון "נעלם" הנמצא ב‬BSA ‫סטנדרט‬
'‫חלק ב‬
‫ מושגים ונספחים‬,‫ רקע‬.3
.‫ הרקע הספרותי נלקח מויקיפדיה‬,‫*אם לא צוין אחרת‬
PBS
Phosphate buffered saline (abbreviated PBS) is a buffer solution commonly used in
biological research. It is a water-based salt solution containing sodium phosphate,
and, in some formulations, potassium chloride and potassium phosphate. The
osmolarity and ion concentrations of the solutions match those of the human body
(isotonic). PBS has many uses because it is isotonic and non-toxic to cells. These
uses include substance dilution and cell container rinsing.
DMEM medium
Eagle's minimal essential medium (EMEM) is a cell culture medium developed by
Harry Eagle that can be used to maintain cells in tissue culture. It contains: salt,
amino acid, glucose and vitamins. A variation of this EMEM, called Dulbecco’s
modiﬁed Eagle's medium (DMEM), contains approximately four times as much of
the vitamins and amino acids present in the original formula and two to four times
as much glucose. Additionally, it contains iron and phenol red. DMEM is suitable for
most types of cells, including human, monkey, hamster, rat, mouse, chicken and
fish cells.
Trypsin
Trypsin is a serine protease from the PA clan (Proteases of mixed nucleophile, superfamily
A) superfamily, found in the digestive system of many vertebrates, where it hydrolyses
proteins. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen.
Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or
arginine, except when either is followed by proline. It is used for numerous biotechnological
processes. The process is commonly referred to as trypsin proteolysis or trypsinisation,
‫ ציטומטר זרימה‬: ‫ניסוי‬
‫ ע"י שחר בן שאול‬:15/02/2015 ‫עדכון אחרון‬
25 ‫ מתוך‬3 ‫עמוד‬
and proteins that have been digested/treated with trypsin are said to have been trypsinized.
In a tissue culture lab, trypsins are used to re-suspend cells adherent to the cell culture
dish wall during the process of harvesting cells. Some cell types have a tendency to "stick"
- or adhere - to the sides and bottom of a dish when cultivated in vitro. Trypsin is used to
cleave proteins bonding the cultured cells to the dish, so that the cells can be suspended
in fresh solution and transferred to fresh dishes.
BSA
Bovine serum albumin (also known as BSA or "Fraction V") is a serum albumin protein
derived from cows. It is often used as a protein concentration standard in lab experiments.
The full-length BSA precursor protein is 607 amino acids in length. An N-terminal 18residue signal peptide is cut off from the precursor protein upon secretion; hence the initial
protein product contains 589 amino acid residues. An additional 4 amino acids are cleaved
to yield the mature BSA protein that contains 583 amino acids.
FBS
Fetal bovine serum (FBS) or fetal calf serum is the blood fraction remaining after the natural
coagulation of blood, followed by centrifugation to remove any remaining red blood cells.
Fetal bovine serum comes from the blood drawn from a bovine fetus via a closed system
of collection at the slaughterhouse. Fetal bovine serum is the most widely used serumsupplement for the in vitro cell culture of eukaryotic cells. This is due to it having a very low
level of antibodies and containing more growth factors, allowing for versatility in many
different cell culture applications. The globular protein, bovine serum albumin (BSA), is a
major component of fetal bovine serum. The rich variety of proteins in fetal bovine serum
maintains cultured cells in a medium in which they can survive, grow, and divide.
GFP
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues
(26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to
ultraviolet range. Although many other marine organisms have similar green fluorescent
proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea
victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm
and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green
portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. In
cell and molecular biology, the GFP gene is frequently used as a reporter of expression.
In modified forms it has been used to make biosensors, and many animals have been
created that express GFP as a proof-of-concept that a gene can be expressed throughout
a given organism. The GFP gene can be introduced into organisms and maintained in their
genome through breeding, injection with a viral vector, or cell transformation. To date, the
GFP gene has been introduced and expressed in many Bacteria, Yeast and other Fungi,
fish (such as zebrafish), plant, fly, and mammalian cells, including human. The availability
of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way
it is used in cell biology and other biological disciplines. While most small fluorescent
molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in
live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated
in living cells. This has triggered the development of highly automated live-cell
fluorescence microscopy systems, which can be used to observe cells over time
expressing one or more proteins tagged with fluorescent proteins.
Fig1. The wide varieties of GFP and variants that have been produced. mRFP1 is an already altered version
of the Discosoma protein. Exc. stands for excitation and is the peak wavelength at which excitation occurs,
while Em. is the peak emission wave length. E before the name of protein signifies enhanced
brightness.[Timothy King and Paul May, University of Bristol]
Trypan blue assay
Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo
dye. Live cells or tissues with intact cell membranes are not coloured. Since cells are very
selective in the compounds that pass through the membrane, in a viable cell, trypan blue
is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are
shown as a distinctive blue colour under a microscope. Since live cells are excluded from
staining, this staining method is also described as a dye exclusion method.
Fig2. A view of trypan blue assay through microscope
Cell line
(Science: cell culture) a cell line is a permanently established cell culture that will
proliferate indefinitely given appropriate fresh medium and space.
Cell lines differ from cell strains in that they have escaped the Hayflick limit and become
immortalized. Some species, particularly rodents, give rise to lines relatively easily,
whereas other species do not. No cell lines have been produced from avian tissues and
the establishment of cell lines from human tissue is difficult. Many cell biologists would
consider that a cell line is by definition already abnormal and that it is on the way towards
becoming the culture equivalent of a neoplastic cell.
J774
A murine macrophages cell line established from a tumor that arose in a female BALB/c
mouse. Its growth is inhibited by dextran sulfate, purified protein derivative, and bacterial
lipopolysaccharides. J774 cells have been used for numerous biochemical studies aimed
at understanding the physiology of monocytes-macrophages.
Normal Human Dermal Fibroblasts (NHDF)
NHDF are isolated from the dermis of juvenile foreskin or adult skin from different
locations like the face, the breasts, the abdomen, and the thighs.
Hybridoma cell line
Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by
fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is
selected for its ability to grow in tissue culture and for an absence of antibody chain
synthesis. The antibodies produced by the hybridoma are all of a single specificity and
are therefore monoclonal antibodies (in contrast to polyclonal antibodies). The use of
monoclonal antibodies is numerous and includes the prevention, diagnosis, and
treatment of disease.
HEK cells
Human Embryonic Kidney 293 cells, also often referred to as HEK 293, HEK-293, 293
cells, or less precisely as HEK cells are a specific cell line originally derived from human
embryonic kidney cells grown in tissue culture. HEK 293 cells are very easy to grow and
transfect very readily and have been widely used in cell biology research for many years.
They are also used by the biotechnology industry to produce therapeutic proteins and
viruses for gene therapy.
Spectrophotometry
Spectrophotometry is one of the most widely used and versatile of all biochemical analytical
tools. It is employed to identify the presence of certain materials in a solution, determine
their concentration, and follow changes of those compounds over time. The analysis of
systems by measuring light absorption has definite advantages.
It is a nondestructive method, unless a photochemical reaction occurs, which is not
common. It offers selectivity, given that each compound has a characteristic spectrum.
Often a particular component in a solution or group in a molecule can be singled out for
observation. It averages the properties of the system over a short time interval, 10 -14 sec
(contrast this with viscosity, which averages the properties of the system over a period of
seconds or minutes), and this enables one to follow the details of fast reactions. Finally,
probably the most important fact is that measurements of high accuracy can be made in a
short time. In general, spectrophotometric measurements characterize absorption /
transmission spectra of materials. This can be done on a wide scale of wavelengths, from
single angstroms to centimeters (see Fig. 12.2). The various types of energy levels in order
of decreasing energy difference are electronic, vibrational, rotational, and spin
orientational (in a magnetic field).
Transitions between electronic levels, promoting to a higher energy electronic
configuration, occur with light in the ultraviolet and visible range. This region of the
spectrum has been and will continue to be of most general use to biochemists, and we limit
our discussion to it. The absorption of visible light - that detected by the human eye - is
most commonly noted by the color of objects. A solution of vitamin B 12, for example,
transmits red light because blue, green, and yellow, the other colors also present in white
light, are absorbed. The spectrum of vitamin B12 is shown in Figure 12.1.
More in general, many materials in nature absorb light. Absorption of light occurs when the
energy of the photon, hν, corresponds to the difference between two energy levels of the
molecule. If the absorbed light is within the visible spectrum, the material appears colored,
where its color is determined by the transmitted light. If the absorption is in the UV region,
the material appears colorless.
A large number of the molecules of biological interest absorb light in the ultraviolet visible
region of the spectrum. Its range in wavelength units is 2000-8000 A0, or 200800 nm. Some examples of biochemical molecules that absorb in this region are
purines, pyrimidines, and aromatic amino acids, and the macromolecules, nucleic acids,
and proteins, which contain residues of them in their structures. Other examples are the
heme-containing proteins (the cytochromes, hemoglobin and myoglobin), and the
carotenoids and steroids. All of these compounds contain conjugated systems which
absorb in the ultraviolet region and some, the cytochromes, hemoglobin and
myoglobin, also absorb in the visible range.
Absorption laws
Spectrophotometry is primarily used by biochemists to monitor the concentration of various
species in reaction mixtures. The absorption of light at a given wavelength is related to the
concentration of absorbering species through two laws. The first, which is attributed to
Bouguer, states that the fraction of light absorbed is proportional to the thickness of the
absorber, x, and independent of the light intensity. Thus for successive layers of absorber
of thickness, x, each absorbs the same fraction of the light incident on it. For example, if
20% of the incident light is absorbed by a thickness, x, the transmitted light is 80%. For
additional thicknesses, x, the transmitted light will be diminished as follows: 64%, 51.2%,
and so on, which is the sequence (0.8)0, (0.8)1, (0.8)2, (0.8)3, and so on. It is expressed
mathematically as:
Where I is the intensity of the transmitted light, I0 is the intensity of the incident light,
'x' is the thickness, and α is the linear absorption coefficient, a constant characteristic of
the medium. Writing in logarithmic form, we obtain
Expression, which is for a uniform solid absorber, can be modified to include solution
concentrations. The path length x is, in a sense, a concentration (uniform solid) as well as
a path length. For a solution, which contains an absorber dispersed in a nonabsorbing
solvent, these terms need to be separated. Light is absorbed only when a photon collides
with a molecule. Thus the amount of light absorbed or the probability of absorption is
proportional to the number of molecules in the light beam. This is a statement of Beer's
law, the second law of absorption. In solution the number of molecules of absorber in the
light beam is proportional to the product of the concentration c and the path length b.
Combining Beer's law with Bouguer's law, we obtain
Where 'a' is the absorptivity or specific absorption coefficient and is characteristic of the
absorbing species (analogous to a). The quantities in the 'log' term are as previously
defined. When the concentration units are moles per liter, 'a' is called the molar
absorptivity or the molar absorption coefficient and is denoted by έ. The absorption law is
also written in terms of absorbance, A, as in
Absorbance (commonly and less desirably called optical density) is then a linear function
of concentration for any given wavelength if Beer's law* is obeyed. The molar absorption
coefficient, however, varies with wavelength and solvent. Beer's law is exact only for
parallel, monochromatic light in an isotropic medium (randomly oriented absorbers in
solution). One should always check to see that it is obeyed. Beer’s law is also limited to a
certain range of concentrations.
* Also known as Beer-Lambert law.
The measurement
The quantities I and I0 for Equations (3-4) are determined for each wavelength as follows.
The intensity of the incident light I0 is obtained from a measurement of a suitable blank
which contains all the solution components except the absorber. Rather than read I0 in
percent, most spectrophotometers enable the reading of log I0 directly.
The transmitted intensity, I, is obtained from a similar measurement on the solution
containing the absorber. If log I is read, then log I / I0 can be obtained by subtraction.
Another term in common usage is transmittance which is the ratio I0 / I.
Automatic scanning spectrophotometers give the absorbance, log I / I0, directly as a
function of wavelength. This continuous comparison is achieved by using two cells, a
sample and a reference, two light beams, and a chopper which enables the detector to
look alternately at the light transmitted by each solution. The concentration of a compound
in a solution can be determined using a calibration curve and beer's law. The calibration
curve is obtained by measuring the absorbance of different solutions with known
concentrations, and its slope denotes the absorption coefficient. The unknown
concentration can then be determined from beer's law. Of course, not all materials in nature
have a specific absorption in the visible/UV spectrum. The concentration of such materials
can often be determined using a chemical or an enzymatic reaction with a colored product.
This concept is utilized in this lab.
The spectrophotometer
This section is also based on: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab3.htm
A spectrophotometer, which is a device for measuring light absorption as a function of
wavelength, has four basic parts: a source of light, the monochromator, the sampling cell,
and the detector (see Figure below). Light from the source, which for the region 200-800
nm is either a hydrogen-discharge lamp or a tungsten bulb, is focused on the entrance slit
of the monochromator, which disperses the light and focuses on the desired wavelength
on the exit slit. This monochromatic light then passes through the sample to the detector.
Fig3.Schema of spectrophotometer components
A typical monochromator contains an entrance slit, a collimating lens, a dispersing device
which can be a prism or a grating, a focusing lens and an exit slit. Polychromatic radiation
(radiation of more than one wavelength) enters the monochromator through the entrance
slit. The beam is collimated, and then strikes the dispersing element at an angle. The beam
is split into its component wavelengths by the grating or prism. By moving the dispersing
element or the exit slit, radiation of only a particular wavelength leaves the monochromator
through the exit slit.
A prism takes advantage of dispersion, which results from the fact that different color of
light travel through glass at different speeds, depending on wavelength. A diffraction
grating can accomplish the same separation of colors because of diffraction. A light ray
reflected (or transmitted) by a groove in the grating will either interfere constructively or
destructively with the ray from the groove next to it, depending on the angle it emerges and
the light's wavelength.
Fig4.Illustration of spectra dispersion
The containers for the sample and reference solution must be transparent to the radiation
which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy
in the UV region. These cells are also transparent in the visible region. Silicate glasses can
be used for the manufacture of cuvettes for use between 350 and 2000 nm. Finally, the
emitted light is collected by a detector. UV-Vis spectrophotometers can feature either
silicon photodiode detectors or photomultiplier tube (PMT) detectors.
1. Scoog, West, Holler, “Fundamentals of analytical chemistry”, 6th ed. (1992), pp.
508-534
2. Segel, I.H., "Biochemical calculations", 2nd. ed. (1976).
3. Montgomery, R. and Swenson, C.A., "Quantitative Problems in the Biochemical
Sciences", 2nd. ed. (1976).
Bradford protein assay
The Bradford protein assay is a spectroscopic analytical procedure used to measure the
concentration of protein in a solution. It is subjective, i.e., dependent on the amino acid
composition of the measured protein. The Bradford protein assay was developed by
Marion M. Bradford. The Bradford assay, a colorimetric protein assay, is based on an
absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic
conditions the red form of the dye is converted into its bluer form to bind to the protein
being assayed. During the formation of this complex, two types of bond interaction take
place: the red form of Coomassie dye first donates its free electron to the ionizable groups
on the protein, which causes a disruption of the protein's native state, consequently
exposing its hydrophobic pockets. These pockets in the protein's tertiary structure bind
non-covalently to the non-polar region of the dye via van der Waals forces, positioning the
positive amine groups in proximity with the negative charge of the dye. The bond is further
strengthened by the ionic interaction between the two. The binding of the protein stabilizes
the blue form of the Coomassie dye; thus the amount of the complex present in solution is
a measure for the protein concentration, and can be estimated by use of an absorbance
reading. The (bound) form of the dye has an absorption spectrum maximum historically
held to be at 595 nm. The cationic (unbound) forms are green or red. The binding of the
dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm
is proportional to the amount of bound dye, and thus to the amount (concentration) of
protein present in the sample.
‫‪The Bradford assay is linear over a short range, typically from 0µg/mL to 2000 µg/mL, often‬‬
‫‪making dilutions of a sample necessary before analysis.‬‬
‫‪It is also inhibited by the presence of detergents (mainly SDS).‬‬
‫‪Much of the non-linearity stems from the equilibrium between two different forms of the dye‬‬
‫‪which is perturbed by adding the protein. The Bradford assay linearizes by measuring the‬‬
‫‪ratio of the absorbances, 595 over 450 nm. This modified Bradford assay is approximately‬‬
‫‪10 times more sensitive than the conventional one (Zor & Selinger, 1995).‬‬
‫‪Centrifuge‬‬
‫צנטריפוגה (בעברית‪ :‬סַ ְר ֶּכזֶּת) היא מתקן להפרדת חומרים‪ ,‬בעיקר חלקיקים זעירים‪ ,‬המרחפים בנוזל‪.‬‬
‫העיקרון הפיזיקלי העומד מאחורי השיטה הוא קיום תלות בין אופן הצטברות החומר בדפנות הצנטריפוגה‬
‫ובין מסת חלקיקיו‪ :‬עם סיבוב הצנטריפוגה‪ ,‬חלקיקים בתוכה נדחפים לעבר הדופן‪ ,‬אך צפיפותם הולכת‬
‫ודועכת ככל שמתרחקים מהדופן ‪ -‬חומרים בעלי צפיפות גבוהה יותר‪ ,‬כלומר כבדים יותר‪ ,‬נעים ומצטברים‬
‫קרוב לתחתית המבחנה‪ .‬לפעולה זו קוראים סרכוז ומשתמשים בה רבות במחקר הביולוגי‪ ,‬על מנת להפריד‬
‫חומרים שונים‪ .‬לדוגמה‪ :‬נעשה שימוש בצנטריפוגה על מנת לבודד ממברנות תאים לבדיקות מסוימות או‬
‫תאים שלמים כחלק מתהליך יצירת תמונת קריוטיפ‪ .‬בתעשייה משתמשים בצנטריפוגות להפרדה של‬
‫מרכיבים בעלי מסה סגולית שונה המעורבבים בתערובת‪ .‬מאחר שחלקיקים שונים הם בגדלים שונים‬
‫ובצפיפויות שונות‪ ,‬כל חלקיק ישקע בכוח צנטריפוגלי מינימלי שונה‪ .‬וכך ההפרדה של הדוגמית לשכבות‬
‫שונות תעשה תחילה על ידי צנטריפוגה של החלקיק הראשוני (למשל תכולת התא לאחר פירוקו) תחת כוחות‬
‫חלשים‪ ,‬סילוק המשקע ולאחר מכן חשיפת התרחיף לכוחות צנטריפוגלים הולכים וגוברים‪ .‬בכל פעם ישוקע‬
‫נתח צפיפויות מסוים לתחתית המבחנה‪ .‬חזרה מושכלת על שיטה זו‪ ,‬תספק שכבות שונות אשר ייצגו חלקים‬
‫שונים של הדוגמית המקורית‪ .‬ניתן גם להרחיף מחדש שכבות מסוימות ששוקעו ולהריצם שוב בצנטריפוגה‬
‫על מנת להגיע לעידון נוסף בהפרדה‪ .‬השיקוע תלוי במסה‪ ,‬בצורה ובנפח הספציפי של המקרומולקולות‪,‬‬
‫בצפיפות הממס‪ ,‬בגודל הרוטור ומהירות הסיבוב‪ .‬מהירות השיקוע יכולה להיות מנוטרת תוך כדי הניסוי על‬
‫מנת לחשב את המשקל המולקולרי‪ .‬קבוע השיקוע (‪ )s‬ניתן גם הוא לחישוב בשיטה זו‪ .‬ערך גבוה של ‪s‬‬
‫יצביע על משקל מולקולרי גבוה‪ .‬כך‪ ,‬חלקיקים דחוסים ישקעו מהר יותר בעוד חלבונים מאורכים בעלי קבוע‬
‫חיכוך גבוה ישקעו לאט יותר‪ .‬בדרך כלל מאפיינים צנטריפוגה לא בקצב הסיבוב שלה אלא בתאוצה שהיא‬
‫מפעילה על הדגם הנבדק‪ .‬זו הבחנה חשובה כי שתי צנטריפוגות שונות בעלות רדיוסי סיבוב שונים‪ ,‬הפועלות‬
‫עמוד ‪ 14‬מתוך ‪25‬‬
‫באותה מהירות סיבוב‪ ,‬יתנו תוצאות שונות כי תפעל בהן תאוצה שונה על המדגם‪ .‬את התאוצה מקובל‬
‫למדוד בכפולות של ‪ - g‬תאוצת הכובד על פני כדור הארץ‪ .‬התאוצה מחושבת ממכפלת הרדיוס במהירות‬
‫הזוויתית בריבוע‪ .‬הוגדר ערך הנקרא "יחסי כוח צנטריפוגלי" (‪ ,)RCF‬והיא מידה של התאוצה המוחלת על‬
‫דגימה מסוימת ונמדדת בכפולות של תאוצת הכובד ונתונה על ידי‪:‬‬
‫‪Microscopy‬‬
‫המיקרוסקופיה בכלל הינה שיטה אשר נועדה לאפשר לאדם לאפיין באמצעות מערכת הראייה עצמים‬
‫זעירים שלא ניתן להבחין בהם בעין בלתי מזוינת ‪.‬מיקרוסקופית האור הנראה בפרט מנצלת את העובדה‬
‫שרוב העצמים בטבע עוברים אינטראקציה כלשהי עם קרני האור הנראה ‪.‬אופי האינטראקציה‬
‫מתבטאבתכונות החומר הנבדק ‪,‬כגון ספקטרום הבליעה ‪,‬מקדם הרפרקציה והצורה הגבישית שלו ‪.‬‬
‫השיטות המיקרוסקופיות מאפשרות תרגום של הבדלים באחד הפרמטרים הנ"ל לתמונה נראית לעין ‪.‬‬
‫איכות התמונה המתקבלת מתבטאת בשני פרמטרים עיקריים – ניגודיות ) קונטרסט ( והפרדה ) רזולוציה(‪.‬‬
‫פרמטרים אלה תלויים בדגימה הנבחנת אך גם בשיטות ההדמיה השונות‪.‬‬
‫עמוד ‪ 15‬מתוך ‪25‬‬
Inverted Fluorescence Microscope
Upright microscope: illumination is below the sample and objective is above.
Inverted microscope: illumination is above the sample and objective is below. It allows
examination of live biological samples which are placed at the bottom of a dish.
Bright field: the light source and the lens are located on two different sides of the sample,
so that only transmitted light is considered. Thus, the image appears dark on a bright
background.
Dark field: the light source and the lens are located on the same side of the sample, so
that only refracted light is considered. Thus, the image appears bright on a dark
background.
Phase contrast: sample contrast comes from interference of different path lengths of light
through the sample.
Condenser: a lens that gathers light from the microscope light source and concentrates it
into a cone of light that illuminates the specimen with uniform intensity over the entire view
field. It is critical that the condenser light cone be properly adjusted to optimize the intensity
and angle of light entering the objective front lens. Care must be taken to guarantee that
the condenser aperture is opened to the correct position with respect to objective numerical
aperture. When the condenser aperture diaphragm is opened too wide, stray light
generated by refraction of oblique light rays from the specimen can cause glare and lower
the overall contrast. On the other hand, when the aperture is made too small, the
illumination cone is insufficient to provide adequate resolution and the image is distorted
due to refraction and diffraction from the specimen.
Numerical aperture: a measure of the lens ability to gather light and resolve fine specimen
detail. Numerical Aperture, or NA, is given by (NA) = n • sin (α). 'n' is the refractive index
of the medium between the lens and the sample (air ~1, water ~1.33, oil ~ 1.5). 'α' is the
angle of the light cone collected by the lens. NA determines the working distance of the
lens, or the distance between the objective lens and the specimen.
Resolution: the ability to see fine details, the shortest distance between two points on a
specimen that can still be distinguished by the observer or camera system. Resolution is
proportional to the wavelength and inversely proportional to the NA: resolution ~ λ/2NA.
Once you can resolve fine details you can magnify them. Every optical system has a finite
resolution; if you magnify objects beyond the resolution the result will be empty
magnification. So, the actual purpose of a microscope is to see small things clearly.
Another, desirable attribute of a microscope is depth of field, which is the range in which
the specimen is at acceptable focus. A microscope that has a thin depth of field will have
to be continuously focused up and down to view a thick specimen. A third feature is that a
microscope has its mechanism for contrast formation. Contrast is the ratio between the
dark and the bright areas. Typically, most microscopes use absorption contrast – the light
wave absorbing areas that are dark. This is called bright field microscopy. There are other
types of microscopes that use more exotic means to generate contrast, such as phase
contrast, dark field and differential interference contrast. The fourth desirable feature is a
strong illumination source. The higher a microscope magnifies the more light will be
required. Powerful illumination can allow additional degrees of freedom when tuning the
microscope components. The illumination source should also be at a wavelength (color)
that will facilitate the interaction with the specimen. The four attributes of an optical system
interact with each other. Resolution and brightness is antagonistic towards contrast and
depth of field. For example, you cannot have maximum resolution and maximum contrast
simultaneously. Theoretically, if you had an infinite resolving system there would be no
contrast to discern the image. It is up to the microscope operator to decide which attribute
is needed to observe in a particular specimen. These relationships are depicted in the
diagram below.
The microscope contains two different groups of interlaced optical planes that are
responsible for controlling illumination and image formation. Collectively, these optical
planes are known as conjugate planes. The first group of planes (termed the aperture
planes) controls the beam path for illuminating light and produces a focused image of the
lamp filament at the plane of the condenser aperture diaphragm, the rear focal plane of
the objective, and the eye point (also called the Ramsden disk) of the eyepiece.
Conjugate planes are in common focus is critical in achieving proper Köhler illumination.
A second set of planes, known as the image-forming conjugate planes include the field
diaphragm, the specimen, the fixed diaphragm of the eyepiece and the retina of the eye
or the surface of a camera detector. By definition, an object that is in focus at one plane
is also in focus at the other conjugate planes of that light path. In each light pathway
(both image-forming and illumination), there are four separate planes that together make
up the conjugate plane set.
http://zeisscampus.magnet.fsu.edu/tutorials/basics/microscopeconjugateplanes/indexflash.html
Epi-Fluorescence Microscopy
Certain molecules, by virtue of their chemical structure, have the ability to emit light of a
specific wavelength following absorption of light of a shorter, higher energy wavelength
(http://www.chemistry.adelaide.edu.au/external/soc-rel/content/jablonsk.htm).
This process of light absorption and re-emission is termed 'fluorescence' and the molecules
which
exhibit
this
behavior
are
termed
'fluorochromes'
(http://pingu.salk.edu/flow/fluo.html). All fluorochromes have characteristic light absorption
and emission spectra. Upon absorption of photons of the excitation wavelength,
fluorochromes become excited into a higher, unstable energy state. This instability is then
restored by the subsequent production of photons of a lower energy emission wavelength.
For example, fluorescein - a commonly used fluorescent dye - absorbs blue light and emits
green light. If a fluorescent dye can be made to interact with specific cellular components
- attached to an antibody that binds to a cellular protein, for example - then it can be used
as a probe for microscopy.
Fig.2. fluorescence image of living cells
A specimen stained with this probe may be illuminated with pure, filtered light
corresponding to its excitation wavelength and then viewed through an emission filter
which is opaque to all other light except for its emission wavelength. The structures tagged
with the fluorescent probe will appear to light up against a black background in a (hopefully)
high contrast image. The light path of a fluorescence microscope is designed to perform
the illumination and the detection described above. All commonly used fluorescence
microscopes utilize 'epi-illumination', which means they illuminate the samples through the
objective lens. This design essentially uses the objective lens as a 'condenser' - providing
efficient illumination and detection at high numerical apertures. Light is produced by a
bright lamp that emits light with spectral peaks corresponding to the fluorescent dyes that
are used. Typically a mercury lamp is used for this because its emission spectra includes
peaks for excitation of the red, green, and blue dyes most commonly used by biologists.
The light is first passed through a heat filter to prevent damage to the specimens, as well
as to the microscope itself. It then passes through an excitation filter which blocks all
incoming light except the excitation wavelengths of the specific dye being examined. This
filtered light then hits a special mirror sitting in an angle of 45º directly above (or below the
objective in the case of an inverted microscope) the objective lens called a dichroic filter.
Dichroics are mirrors that have special coatings which make them reflective to certain
wavelengths and transparent to other, longer wavelength light. The excitation light is
reflected from the dichroic mirror by an angle of 90º, through the lens, and onto the
specimen. If any fluorescent dye in the field becomes excited, its emitted light is collected
by the objective and passes through the dichroic mirror. Emitted light then passes through
a third emission filter (barrier) which absorbs all light except for the emitted wavelength.
This last filter ensures that any stray excitation light does not contribute to the final image.
Once passed through the barrier filter, the distribution of the dye within the sample is
registered on your eye or on a camera, producing an image of its distribution. Typically
each class of dye (blue, green, or red) has its own set of excitation, dichroic, and emission
filters which work together and are associated as a 'cube'. Different cubes are selected by
moving a slider or rotating a wheel within the body of the microscope.
‫‪ .4‬דו"ח מכין‬
‫ענו בקצרה על שאלות אלו כחלק מהכנה למעבדה ובססו את תשובותיכם על בסיס חומר הרקע‬
‫אשר ניתן לכם בחלק הקודם‪ .‬עם זאת‪ ,‬במקרה והנכם מרגישים כי החומר המוצג אינו מספיק דיו‬
‫להבנת השאלות‪ ,‬היעזרו בקישורים הנוספים המכוונים לאתרי האינטרנט השונים או בצעו חיפוש‬
‫אינטרנטי על פי שיקול דעתכם‪ .‬חלק מהשאלות הינו מחשבתי ועל כן אין בהכרח הורדת נקודות על‬
‫מתן תשובה לא מדוייקת כל עוד הראתם יכולת חשיבה ומאמץ אחר התשובה הנכונה‪.‬‬
‫‪ .1‬מהו טריפסין? למה משמש אותנו בתרביות תאים וכיצד?‬
‫‪ .2‬מהו חלבון ה‪ ?GFP-‬מהו אורך הגל בשיא העירור ומהו תחום הפליטה שלו? אלו יתרונות יש‬
‫בחלבון זה על פני חלבונים פלורוסנטים אחרים אשר פולטים בספקטרום דומה? אלו עוד‬
‫מולקולות\ חלבונים פלורוסנטים יש בנמצא?‬
‫‪ .3‬כיצד ניתן להבדיל בין תאים חיים למתים במבחן על בסיס ‪?trypan blue‬‬
‫‪ .4‬מהו קו תאים? מהיכן מקורו? האם סוג התאים הינו סרטני או נורמאלי?‬
‫‪ .5‬מהו מקור התאים (‪,HEK ,Normal Human Dermal Fibroblasts (NHDF‬‬
‫‪j774‬‬
‫)‪ ?Hybridoma(12Ca5‬מה תפקידם של קווי תאים אלו?‬
‫‪ .6‬תאר בצורה סכמטית את מנגנון הפעולה של מכשיר הספקטרופוטומטר‪.‬‬
‫‪ .7‬מהו ספקטרום הבליעה הטבעי של רוב החלבונים? מהו ספקטרום הבליעה בשיטת ‪?Bradford‬‬
‫מדוע יש הבדלים?‬
‫‪ .8‬מהם הפרמטרים המשפיעים על שיקוע חלקיקים בעת תהליך הסירכוז?‬
‫‪ .9‬נתונים לי ערכי ‪ RCF‬ו ‪ RPM‬של צנטריפוגה מסוימת לשם שיקוע תאים‪ .‬כאשר אעבור‬
‫לצנטריפוגה אחרת‪ ,‬מי מהערכים עלול להשתנות ומי יישאר רלבנטי לשם שיקוע התאים?‬
‫‪ .10‬מהו מיקרוסקופ הפוך (‪ ,)inverted‬ומה היתרון בשימוש בו?‬
‫‪ .11‬מהם ארבעת המאפיינים העקריים במקרוסקופ וכיצד הם משפיעים אחד על השני?‬
‫‪ .12‬תאר את מסלולה האופטי של קרן אור במיקרוסקופ פלורוסנטי‪.‬‬
‫עמוד ‪ 22‬מתוך ‪25‬‬
‫חלק ג'‬
‫תיאור מהלך מערכת הניסוי‬
‫‪.5‬‬
‫‪‬‬
‫סקירת מכשירי המעבדה‪ :‬מנדפים ביולוגים וכימיים‪ ,‬פסולת ביולוגית וכימית‪ ,‬פיפטורים‬
‫מכאנים וחשמליים‪ ,‬צנטריפוגות שונות‪ ,‬אפנדורפים ומבחנות‪.‬‬
‫‪‬‬
‫תרגול פיפטציה עם הפיטורים השונים‪.‬‬
‫‪‬‬
‫הסבר על חומרים ותמיסות‪ :‬מדיום‪ ,‬סרום‪ ,‬מים מזוקקים‪ ,‬תמיסות מלח לניסויים בתאים‬
‫וריאגנטים לצביעת תאים‪.‬‬
‫‪‬‬
‫סקירה תיאורטית של ארבעת קווי התאים השונים‪.‬‬
‫‪‬‬
‫הסתכלות במיקרוסקופ אור‪.‬‬
‫‪‬‬
‫הסתכלות בתאים בעזרת מיקרוסקופ פלורסנטי ודרך מצלמה‪..‬‬
‫‪‬‬
‫מיצוי תאים מצלחת‪:‬‬
‫‪.i‬‬
‫שימו כפפות‪ ,‬הכינו את הפיטורים השונים וסמנו ‪ 4‬מבחנות ‪15ml‬עם שמות קווי התאים‬
‫השונים (כל מבחנה עבור קו תאים אחר)‪.‬‬
‫‪.ii‬‬
‫שאיבת מדיום מהצלחות השונות‪:‬‬
‫‪ )a‬שאבו את המדיום מהצלחות ‪ j774‬ו ‪ HEK‬ומלאו במקום ‪ 2ml‬של ‪.PBS‬‬
‫‪ )b‬שאבו את המדיום מהצלחת של תאי ‪ ,NHDF‬ובצעו שטיפה בעזרת ‪.PBS 2ml‬‬
‫לאחר הוצאת ה ‪ PBS‬הוסיפו ‪ 2ml‬טרפסין והכניסו את הצלחת ל‪ 3-‬דקות‬
‫באינקובאטור‪.‬‬
‫‪ )c‬על מנת למצות את תאי ההברידומה יש לאסוף את המדיום עם פיפטה ולהכניסו‬
‫למבחנת ‪.15ml‬‬
‫‪.iii‬‬
‫גרדו בעזרת ‪ cell scraper‬את תאי ה‪ j774 -‬והעבירו למבחנת ‪.15ml‬‬
‫‪.iv‬‬
‫תאי ה‪ HEK-‬יוסרו בעזרת פיפטציה של ה‪ PBS -‬על גבי הצלחת ולאחר מכן העברתם‬
‫למבחנת ‪.15ml‬‬
‫‪.v‬‬
‫בדקו את הצלחת עם תאי ה‪ NHDF-‬אשר באינקובאטור‪ .‬אם התאים מרחפים בצלחת‬
‫יש להוסיף ‪ 8ml‬של מדיום ולהעבירו למבחנת ‪.15ml‬‬
‫‪.vi‬‬
‫יש לוודא כי כל המבחנות מאוזנות בנפח זהה ‪.‬סרכזו את התאים ב ‪ 200rcf‬למשך ‪5‬‬
‫דקות‪.‬‬
‫עמוד ‪ 23‬מתוך ‪25‬‬
‫‪.vii‬‬
‫הוציאו בזהירות את המבחנות מהצנטריפוגה‪ ,‬וודאו כי אכן ישנו משקע של תאים‪ .‬הסירו‬
‫נוזל עליון והרחיפו תאים ב‪ 1ml -‬של ‪.PBS‬‬
‫‪‬‬
‫ספירת התאים ובדיקת חיות‪:‬‬
‫‪.i‬‬
‫הוסיפו ‪ 20µl‬של טריפן ‪ Trypan blue‬אל מבחנת אפנדורף‪.‬‬
‫‪.ii‬‬
‫הוסיפו ‪ 20µl‬של התאים המרחפים אל תוך מבחנת האפנדורף‪.‬‬
‫‪.iii‬‬
‫ערבבו היטב את התמיסה ולאחר מכן המתינו כדקה‪.‬‬
‫‪.iv‬‬
‫הכינו את ההמוציטומטר עם זכוכית המכסה‪.‬‬
‫‪.v‬‬
‫קחו ‪ 10µl‬של התמיסת צבע עם התאים והזריקו בין הזכוכית לתא הספירה ותנו לנוזל‬
‫להישאב פנימה‪.‬‬
‫הסתכלו על השרטוט במיקרוסקופ וסיפרו את מס' התאים הצבועים והלא צבועים‪.‬‬
‫חשב את כמות התאים על סמך הנוסחא‪:‬‬
‫‪.vi‬‬
‫‪.vii‬‬
‫‪ ‬יצירת עקומת כיול לכימות חלבונים‪:‬‬
‫‪ .i‬הכינו ‪ 9‬קווטות עם ריאגנט ‪ 1ml‬של ‪.Coomassie blue‬‬
‫‪.ii‬‬
‫הוציאו מבחנת ‪ BSA 2mg/ml‬מהמקרר והזריקו נפח של חלבון בסדר עולה ‪(2.5µl,‬‬
‫)‪5µl, 10µl, 20µl, 40µl‬‬
‫עמוד ‪ 24‬מתוך ‪25‬‬
‫‪.iii‬‬
‫בשלושת המבחנות האחרות הזריקו ‪ 20µl‬של ‪ 10% FBS‬וכן ‪ 20µl, 40µl‬של מדיום‬
‫עם סרום ו‪ 20 µl -‬ממבחנת ה"נעלם"‪.‬‬
‫‪.iv‬‬
‫קראו במכשיר הספקטרו‪-‬פוטומטר את כל הקווטות‪.‬‬
‫‪.v‬‬
‫מצאו ריכוז חלבון כללי בכל אחת מהקווטות‪.‬‬
‫‪ .7‬דרישות הדו"ח המסכם‬
‫הדו"ח צריך היות קצר וענייני‪ .‬יש להגישו לא יאוחר משבועיים מיום ביצוע המעבדה‪ .‬על כל עיכוב‬
‫יש להודיע מראש‪ ,‬כל יום איחור ילווה בירידת ‪ 5‬נקודות מהציון‪.‬‬
‫אין צורך לרשום את הפרוטוקול עבודה בשנית‪.‬‬
‫‪ .1‬רשמו תקציר קצר המתארת את מטרות ורקע כל ניסוי‪.‬‬
‫‪ .2‬כתבו כיצד חשבתם את ריכוז התאים‪ ,‬חיות התאים‪ .‬אילו עוד פרמטרים עשויים להשפיע על‬
‫הריכוז הסופי וחיות התאים?‬
‫‪ .3‬הכינו והציגו את עקומת הכיול בתוכנת ‪ .excel‬שימו לב לסמן את צירי הגרף ביחידות הנכונות‪.‬‬
‫‪‬‬
‫חשבו את ריכוז שלושת המבחנות ‪ FBS‬ומהן הסיקו מהו ריכוז החלבון של מבחנת המקור‪.‬‬
‫‪‬‬
‫חשבו את ריכוז מבחנת הנעלם‪.‬‬
‫עמוד ‪ 25‬מתוך ‪25‬‬

ניסוי: הכרת מכשור מעבדתי

Transcription

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ה ״ ע ש ת ט ב ש ,1 ס׳ מ ן ו י ל י ג , ב ״ ל ך ר כ