Immunology Methods for
Medical Students
(Second Edition)
Department of Immunology
China Medical University
2007
September，2007
September
1
Preface
IMMUNOLOGY METHODS FOR MEDICAL STUDENTS is
a reference and guide booklet for medical students to refer to
when doing lab practice of their immunologic skills. It
includes some basic classic immunologic experiments as
well as some advanced techniques that frequently used in
research work. These experiments would help students to
achieve a better understanding of immunologic theories. The
writers of this booklet are Li Zongxi, Zheng Li, Feng Hui,
Cao Yan, Li Cheng and Pang Wei.
Department of Immunology
Nov 11, 2006
2
Exp. I Agglutination Reaction……………………………..……….1
i. Tube Agglutination Reaction…………………………….. 1
ii. Slide Agglutination Reaction…………..………….….…….4
iii. Indirect Agglutination Inhibition Reaction………….……..6
Exp. II Precipitation Reaction …………..……………………….7
i. Double Diffusion Reaction……….……………………….7
ii. Single Diffusion Reaction.……….…………………………9
Exp. III Erythrocyte Rosette-forming Cell Test ……………………13
Exp. IV Enzyme-Linked Immunosorbent Assay……………………16
Exp. V Separation of Murine T, B lymphocyte…….…….……...19
i. Preparation of murine splenolymphocytes……………..….19
ii. Preparation of murine thymocytes……………..………….19
Exp. VI Detection of T Lymphocytes Subgroup………………..…21
Exp. VII Measurement of Phagocytosis by Phagocytes ………..….24
3
Exp. I Agglutination Reaction
Agglutination Reaction includes:
1. Direct agglutination reaction: tube agglutination reaction and slide
agglutination reaction.
2. Indirect agglutination reaction.
i. Tube Agglutination Reaction
[Principle]
Agglutination is the interactions between insoluble particles (e.g. intact bacteria
and cells) or soluble antigens that attached to particles and their corresponding
specific antibodies that result in some visible agglutinates if given enough time
and the proper concentration of electrolyte.
Tube agglutination test is usually used in the semi-quantitative test, by mixing the
known particle antigen directly with serial dilutions of diagnostic serum in the
tubes. Observe the appearance of agglutinates in the tubes after certain time.
Determine the level of the antibody in the serum and its titer according to the
relative agglutination amount.
[Application]
A classic application of tube agglutination test method is the Widal test, a
diagnostic procedure for detection of Salmonella infections, where the presence
of antibodies against Salmonella H and O antigens can be demonstrated in the
patients’ serum. It can help to diagnose Typhoid fever.
This experiment is to determine the antibody titer of rabbit anti-sheep-erythrocyte
serum using sheep erythrocytes. The titer is the reciprocal of the last dilution of an
antiserum capable of mediating some measurable effect such as precipitation or
agglutination. It is a measure of the relative strength of an antiserum.
[Materials]
1. 1% fresh sheep erythrocyte;
2. Rabbit anti-sheep erythrocyte serum (heat inactivation of complement at 56
℃ for 30 min);
4
3. Saline, tube, pipette, test tube rack, etc.
[Procedures]
1. Put eight tubes in the rack and label them from 1 to 8.
2. Add 0.5ml of saline to each tube using the 1ml pipette.
3. Add 0.5ml of rabbit anti-sheep erythrocyte serum (1:10 diluted) to tube 1,
mixed by sucking and pumping the fluid with pipette for 3 times.
4. Transfer 0.5ml of dilution from tube 1 to 2, mixed well as above. Then transfer
0.5ml of dilution to tube 3. Repeat the same procedure in tube 3,4,5,6 and 7 for
serial dilution of serum and discard 0.5ml of dilution from tube 7. Notice: don’t
do the same to tube 8. Tube 8 is set as negative control without serum mixed in.
The dilution rate of serum from tube 1 to 7 now is 1:20, 1:40, 1:80, 1:160,
1:320, 1:640, 1:1280 (Fig 1-1).
5. Add 0.5ml of sheep erythrocyte to each tube, mix well by shaking gently. The
final dilution of serum from tube 1 to tube 7 now is 1:40, 1:80, 1:160, 1:320,
1:640, 1:1280, 1:2560.
6. Transfer the tubes into the racks in water bath. Incubate for 3 hours at 37℃ (or
overnight in incubator at 37℃).
Fig 1-1 Serial dilution of rabbit anti-sheep erythrocyte serum
[Resullts]
[Resu
1. Take out the tubes from the water bath and do not shake the tubes when
observing the results. First observe the control tube 8. There is no agglutination
in the control tube 8. It shows a red dot at the bottom of the tube result from
erythrocyte sedimentation. Then observe the results from tube 1 to tube 7. The
agglutination in the bottom of the tubes represents positive reaction. The
agglutination degrees are represented with (++++), (+++), (++) and (+)
5
respectively.
2. Assessment standards:
1
○
(++++): represents all of the sheep erythrocytes agglutinated. A big piece of
agglutinant appears uniformly at the bottom of tubes and agglutinant brim
crimpled as petal, while the supernatant is clear.
2
○
(+++): represents about 75% sheep erythrocytes agglutinated. A piece of
agglutinant appears. The area of agglutinant is bigger than (++), while the
supernatant is a little turbid.
3
○ (++): represents about 50% sheep erythrocytes agglutinated. The area of the
agglutinant becomes smaller and agglutinant brim becomes looser, while half
of the supernatant is turbid.
4
○
(+): represents about 25% sheep erythrocytes agglutinated. The erythrocyte
sedimentation at the bottom of the tube is surrounded by a little of agglutinant.
[Attentions]
1. Gently manipulate the pipettes. Do not break the test tubes with the pipettes.
Adding and transferring the samples one by one to avoid confusion.
2. Do not shake the test tubes when you observe the results, to avoid disturbing
the agglutinants during observation.
6
ii
ii.. Slide Agglutination Reaction
[Principle]
Slide agglutination reaction is the direct agglutination carried out on the slides, by
directly mixing the antibody with a particle antigen under the certain
concentration of electrolyte. The result is positive when there is visible
agglutinate, otherwise negative.
[Application]
Slide agglutination is a qualitative test. It may be used mostly to determine blood
types (ABO types) or if antibody to bacteria is presented in blood as an indication
of infection of the bacteria. This experiment is to determine ABO blood types.
[Materials]
1.
2.
3.
4.
Human peripheral blood
Standard sera: Anti-A and anti-B sera
Saline
Slides, needles, cotton ball, etc.
[Procedures]
1. Slides preparation: Take a clean slide from the bottle, wipe clean and dry out.
Plot it into three squares and label as “1”, “2”and “3”.
2. Add a drop of anti-A and anti-B standard serum on square “1” and square “2”,
respectively. Add a drop of saline on square “3”. (Fig.1-2)
3. Sting finger tip with sterilized needle, take blood using brim of a sterilized slide
from the box and mix the blood into the sera and saline on square “1”,“2” and
“3”, respectively.
4. Shake slide gently, mix blood and sera well and observe the results in 1~2 min.
1
anti-A
2
anti-B
3
saline
Fig 1-2. Blood type determination
7
[Results]
Positive result shows particle agglutination. Negative result appears uniformly
turbid. You can observe the results by microscope if the results are not clear.
[Results Analysis]
Blood
type( phenotype)
A
B
O
AB
Antigen on RBC
Antibody in sera
Gene type
A
B
H
A, B
Anti-B Ab
Anti-A Ab
Anti-A Ab, Anti-B Ab
----
A/A, A/O
B/B, B/O
O/O(H)
A/B
[Attentions]
1. Slides require to be sterilized in 75% ethanol.
2. Take blood with different sides of the brim of slide.
8
iii
iii.. Indirect Agglutination Inhibition Reaction
[Purpose]
Pregnancy determination (Human Chorionic Gonadotropin, HCG detection in
urine)
[Materials]
1.
2.
3.
4.
5.
Urine samples ( with HCG)
Standard HCG immunized latex granule
Standard Anti-HCG Ab
Saline
Black glass board
[Methods]
1. One drop of Ab on each of the 3 squares.
2. Sample 1st, 2nd and saline in corresponding square, shake, react for 1 min.
3. One drop of immunized latex granule in each square, shake, react for 4 to 7
min.
[Result observation]
Sandy agglutination in sample 2 and saline
[Conclusion]
Agglutination (+) HCG (--) Pregnancy (--)
Agglutination (--) HCG (+) Pregnancy (+)
[Questions]
What are the applications of agglutination test?
What is titer?
How the antigen and antibody attach to each other?
9
Exp. II
Precipitation Reaction
i. Double Immunodiffusion Reaction
[Principle]
Precipitation reaction occurs when soluble antigen (serum proteins, cell lysate or
tissue lysate) binds its specific antibody and produce visible precipitate.
In Double Immunodiffusion Test, the solution of soluble antigen and antibody are
added into corresponding wells in the agar plate respectively, then let them diffuse
freely. When the antigen meets with its corresponding antibody, they will combine
with each other. If the conditions (pH, temperature and concentration) are
appropriate, there is a band of precipitate composed of the specific antigen and its
antibody. The characteristics of antigen and antibody can be analyzed according to
the existence of precipitation band, as well as its location and shape.
[Application]
It’s mostly used to analyze the characteristics of antigen or antibody, determine
the purity of antigen and antibody or measure the titer of antibody. This test is to
detect α-fetoprotein (AFP) in the serum.
[Materials]
1. 1.5% saline agar (1.5% agar melted in 0.9% saline solution)
2. Antigen: sample serum, AFP positive serum from patients with liver cancer (or
umbilical cord blood)
3. Antibody: AFP diagnostic serum (anti-AFP antibody)
4. Plastic plate (2.7cm×7cm), agar-puncher, microsyringe, humid box, etc
[Procedures]
1. Preparation of agar plate: 3.5ml melted saline agar solution is poured into a
plastic plate on the horizontal table. 7 wells with diameter of 3mm are punched
in the solidified agar with 5mm of interspace to each other. The pattern of wells
looks like a plum blossom.
10
2. Adding samples: sample serum, AFP positive serum and AFP diagnostic serum
are respectively added into three wells by microsyringe, about 15μl in each well
(clean the microsyringe 3 times with distilled water when changing samples).
(Fig 2-1)
Fig 2-1
3. Put the plate into a humid box and incubate for 24h at 37℃.
[Results]
The result is determined by the existence or nonexcistence of characteristic white
band of precipitate. If an antibody is specific to the corresponding antigen, there is
a complete fusion precipitate band between them. If two precipitate bands cross
each other when they meet, it indicates that two antigens are completely different
and so are the antibodies. If the precipitate bands accord partially with each other,
this type of reaction might indicate the antibody is a kind of mixture antibodies
and unknown antigen is partly identical with control antigens (Fig 2-2).
Fig 2-2
[Attentions]
11
1. When prepare the agar plate, pour the melted saline agar solution into a plastic
plate rapidly, for the melted agar will solidify quickly.
2. Before punching the actual wells, practice at the corner of the agar plate.
3. Don’t lacerate the agar while punching wells and adding samples, in order to
avoid any influence on the formation of the precipitate band.
[Question]
What are the applications of the double immunodiffusion technique?
ii.. Single Immunodiffusion Reaction
ii
[Principle
Principle]]
A melted agar solution mixed with specific antibody is poured into the plastics
plate. Then wells are punched and filled with known concentration of standard
antigen which can diffuse into the agar and react with the antibody. When the
antigen diffuses to a suitable concentration, it will react with the antibody and
ring-shaped bands of precipitate will be formed concentrically around the well.
It’s advisable to employ a series of dilutions of a standard antigen as a reference,
because the diameters of the precipitation rings show a linear relationship to the
concentration of the standard antigen. The standard curve can be obtained by
using the diameter as ordinate and the dilution rates as abscissa. The concentration
of the sample antigen can be calculated based on the diameter of its precipitation
ring according to the standard curve.
[Application
Application]]
IgG, IgM, IgA, complement, transferrin, antitrypsin, glycoprotein, prealbumin and
many kinds of serum can be measured by this technique quantitatively. In this
experiment, we will detect the concentration of human IgG in sample serum.
[Materials
Materials]]
1. Antigen: the tested human serum, the known concentration of IgG as reference
protein (11.52mg/ml).
2. Antibody: anti-human IgG antibody mixed into 3.3% saline agar solution
12
4. Pipette, 2.7cm×7cm plastic plate, puncher (D=3mm), microsyringe, humid box,
and semi-logarithm coordinate paper.
[Procedures
Procedures]]
1. Preparation of agar plate:
1 3% saline agar is melted by boiling water. Then keep it at 56℃.
○
2 Anti-human IgG is diluted properly by 0.9% saline solution (dilution degree is
○
determined by the titer of antibody).
3
○ Mix agar with equal amount of diluted IgG antibody. Then pour solution into
plastic plates immediately and a 2 mm thick layer of 1.5% agar are made.
2. Dilutions of reference and sample serum:
1
○
0.5ml distilled water is added into every freezing control serum. After
complete dissolving, the serum is diluted into a series of different dilutions. For
example: the concentration of control serum IgG is 11.52mg/ml. Dilutions of
reference serum: 1:18, 1:36, 1:72, 1:144, 1:288. Concentration of
corresponding IgG (μg/ml): 640, 320, 160, 80, 40
2
○ Sample serum is diluted into 1:40 by saline.
3. Punching wells: wells are punched with agar puncher on the antibody-agar after
its solidification according to figure 2-3. The distance between adjacent wells is
about 15mm.The agar is picked up from wells by needle. Be carefully about the
edge of well.
4. Adding samples: Around 10-15μl reference serum and sample serum is added
into agar wells with different dilutions. Make sure the exact same amounts of
samples were added in each well on the same agar plate.
5. Reaction: the prepared plates are incubated in a humid box at 37℃ for 24~48h.
[Results
Results]]
1. Take out agar plate, you will find milky white precipitate ring. Measure the
diameter and write it down (Fig 2-3).
13
Fig 2-3
2. The reference curve: Measure the diameters of precipitin rings and draw a
standard curve with dilution rates as ordinate and the diameters of rings as
abscissa (Fig 2-4).
precipitate ring
diameters(mm)
Dilution rates
15
13
11
9
6
18
36
72
144
288
Dilution
350
Dilution
300
Expon. (Dilution)
250
200
150
y = 2103.8e - 0 . 3 1 2 5 x
100
50
0
0
5
10
15
20
Diameter
Fig 2-4
3. Measure the IgG concentration of the sample serum: Measure the diameter of
the precipitate ring of the sample and the concentration of sample IgG can be
calculated from the equation.
[Attentions
Attentions]]
1. This experiment belongs to quantitative test and could be influenced by many
effect factor, for example, the quality of reference serum, the concentration of
antibody, the quality and concentration of agar, the thickness and uniform of the
agar layer。 So it must be strictly controlled.
2. The reference curve can only be used once but not in other tests.
14
3. While mixing antibody into agar, the temperature of the agar solution shouldn’t
be too high in order avoid denaturalization and inactivation of the antibody.
And it shouldn’t be too low in order to avoid the agar solidify before spreading
out on the plate.
4. The samples must be add accurately and to avoid the alveolus formation in the
wells.
5. The diameter must be checked accurately. If not, the error will be expanded for
several times when multiplying by the dilution rates.
[Question
Question]]
What are the principle and the application of single immunodiffusion technique?
15
Exp. III
Erythrocyte Rosette-forming Cell Test, ERFC
[Principle]
The surface of T lymphocytes contains the receptor (CD2) of sheep erythrocyte,
which is called E receptor for short. Therefore, T lymphocytes can form E rosette
with sheep erythrocytes by its E receptor. The number of E rosettes can imply T
lymphocytes’ number. This test is used to detect the number of T lymphocytes
from peripheral blood.
[Application]
Separation of peripheral blood mononuclear cells (PBMC) and counting of T
lymphocytes in vitro.
[Materials]
1.
2.
3.
4.
5.
6.
7.
Human peripheral blood
Heparin solution, 500U/ml
Lymphocyte separation medium(LSM), specific gravity 1.077±0.001g/L
2% sheep erythrocyte
Ca2+ and Mg2+ free Hank’s balanced salt solution (HBSS)
0.5% methene blue staining solution
Centrifuge tube, test tube, pipette, waterbath, centrifuge, microscope, etc.
[Procedures]
1. Preparation for lymphocyte suspension
1
○
Draw 2ml human peripheral blood. Place into a test tube containing 0.1ml of
heparin and add equal volume of HBSS. Mix well. Pipe 1ml LSM into a
centrifuge tube. Carefully overlay the LSM with 1ml diluted blood sample by
slowly adding blood along the inner wall of test tube (Fig 3-1). The surface of
the LSM must not be disturbed.
16
2
○
3
○
Fig 3-1
Centrifuge at 2000r/min for 20min at room temperature.
Using a pipette, discard the plasma layer and harvest the mononuclear cell
band from tube at the interface between the plasma and LSM. Transfer the
mononuclear solution to a clean test tube containing 4ml HBSS (Fig 3-2).
Mix well.
Fig 3-2
4
○
Wash the cell by centrifuge at 1500r/min for 10 min. Discard the supernatant.
The cell left is mononuclear cell.
2. Add 0.2ml 2% sheep erythrocyte suspension to the test tube with the
mononuclear cell. Mix gently. Place in 37℃ waterbath for 10 min. Then
centrifuge at 1000r/min for 10min and incubate at 4℃ for 40min.
3. Discard the supernatant. Add a drop of 0.5% methene blue. Mix gently. One
drop of solution is aspirated and transferred to a microslide. Put a cover glass.
Count the rosette forming cells under the microscope.
[Results]
The percentage of E-rosette formation is calculated by counting the rosette
forming cells from 200 viable lymphocytes. The lymphocyte is counted as a
17
rosette forming cell if three or more erythrocytes adhere to it.
[Attentions]
1. Blood must be used within 4 hour after collection.
2. Suitable ratio between sheep erythrocyte and lymphocyte should be around
100:1.
3. Do not shake the test tube after E-rosette formation to prevent the rosettes from
falling apart.
[Question
[Questionss]
What is the principle of E-rosette formation?
How is the lymphocytes separated from the other ingredients of blood?
18
Exp. IV Enzyme-Linked Immunosorbent Assay (ELISA)
[Principle]
The principle of ELISA is based on the high specificity of antigen-antibody
reactions. It keeps immunogenic and activities after antigen or antibody absorbs to
the surface of solid-phase. ELISA may be quantified by determining with a
spectrophotometer the rate at which the enzyme converts a clear substrate to a
colored product. The assay is called enzyme-linked immunosorbent assay. ELISA
is now widely used in many areas in the medicines.
Fig. 4-1 Principles of enzyme-linked immunosorbent assay (ELISA)
19
There are many different approaches to ELISA (Fig.4-1). They are typically
performed as a direct or an indirect method, but can also be performed as a two
antibodies sandwich method and a competitive assay. Here we introduce
sandwich ELISA.
[Application]
ELISA is a very sensitive laboratory method and can be quantitative when used in
conjunction with standard curves. It can quantify the amount of antigen or
antibody present in a given sample.
[Purpose]
Double Abs sandwich ELISA detecting HBsAg in serum.
[Materials]
1. [Test Kits]
1) HRP-labeled Ab 2
2) Positive control
3) Negative control
4) Concentrated eluant
5) Chromogenic substrate A
6) Chromogenic substrate B
7) Termination solution
8) Ab 1 coated wells
9) Tape
2. Sample 1, 2, 3
3. Distilled water
4. Micropipettor and tips
5. Pipette
6. Beaker
7. Measuring cylinder
8. Absorbent paper
[Procedures
Procedures]]
20
1. Add samples, 1 row of 12 wells for each 2 or 3 students. Pipet 50l of each
of these 5 elements--3 samples, the positive control and the negative
control--into 2 wells. The empty 2 wells are called blank. We will used
these blank wells later for adjusting the microplate reader. Caution: Slowly
drawing fluid, you should empty the pipettor to the second stopping point,
adding the samples slowly.
2. Pipet 50l of HRP marked Ab in each of the 10 wells you used. Mix, tape,
label and incubate at 37℃ for 30 min.
3. Preparation of eluant: For 1:20 dilution, each table of students should pipet
2ml of eluant and pour 38ml of distilled water into the beaker.
4. Discard the liquid in the well, dry the outside of the wells with filter paper,
and wash the wells with the diluted eluant. Caution: To prevent overflow
don’t add too much eluant to any of the wells. Wait for 5-10 seconds before
discarding the eluant from the wells. Repeat washing the wells 5 times.
5. Add 50l of chromogenic solution A into all 12 wells, and then add
solution B. Mix, tape, label and incubate at 37℃ for 15 minutes.
6. Terminate the reaction with 50l of termination solution in each well.
7. Read the plate at 450nm for OD values
[Result analysis]
Cutoff value: COV= mean value of negative control OD values×2.1
Sample OD>=COV is positive, < COV is negative
Lowest negative mean OD value is 0.05
[Attention]
1.
2.
3.
4.
5.
6.
7.
Samples and test kit need to be used under room temperature
Shake reagents before using
Slowly draw and discharge samples with micropippettor
Change tips between solutions
Filter paper and tape can’t be reused
Wash wells carefully to prevent overflow
Plate needs to be read within 10 minutes after adding the termination
solution
[Question]]
[Question
What are the principles of ELISA?
21
Exp. V Separation of Murine T, B lymphocytes
i. Preparation of murine splenolymphocyte
splenolymphocytess
[Materials]
1.
2.
3.
4.
5.
6.
BALB/c mouse
RPMI 1640, gentian violet
Sterilized culture dishes, scissors, forceps
Sterilized stainless steel screen (200-mesh)
pipettes, tip, EP tube, syringe
Centrifuge, light microscopes
[Procedures]
[Procedures
1. A mouse is sacrificed. Put the mouse into 70% alcohol for 10 minutes. Fix the
mouse lying on its abdomen on a board. Cleave its left back skin and expose
the spleen. Then take out its spleen sterilely and put it in a plate.
2. Put the mouse spleen on a stainless steel 200-mesh screen in a dish.
3. Add 1ml serum-free RPMI-1640 medium to the dish, Pound the spleen into
pieces with forceps. Collect the suspension without dregs or filter the mixture
with gauze. The collected solution is centrifuged at 1,500 r/min for 10 min.
4. The supernatant is discarded. Lyse RBCs by adding 3 ml sterile distilled water
into the tube and stand for 40s. Then add 1 ml 3.6% saline solution or resume
isotonic state and stop lysis. Then resuspend cells.
5. Centrifuge cell suspension at 2,000r/min for 10 min and discard supernatant.
Cells are resuspended and adjusted to needed concentration with culture
medium.
6. Counting the cell, and dilute the cell 107/ml.
ii
ii.. Preparation of murine thymocyte
thymocytess
[Materials]
1. RPMI 1640
2. Cell culture flasks, pipettes, 96-well plates
3. Centrifuge, light microscopes
22
[Procedure
[Proceduress]
1. A mouse is sacrificed. Put the mouse into 70% alcohol for 10 minutes. Fix the
mouse lying on its abdomen on a board. Cleave its left back skin and expose
the spleen. Then take out its spleen sterilely and put it in a plate.
2. Pound the spleen into pieces with forceps. The put the spleen into a tube with
2ml culture medium for 5min. Collect the suspension without dregs or filter
the mixture with gauze. The collected solution is centrifuged at 1,500 r/min
for 10 min.
3. The supernatant is discarded. Lyse RBCs by adding 3 ml sterile distilled water
into the tube and stand for 40s. Then add 1 ml 3.6% saline solution or resume
isotonic state and stop lysis. Then resuspend cells.
4. Centrifuge cell suspension at 2,000r/min for 10 min and discard supernatant.
Cells are resuspended and adjusted to needed concentration with culture
medium.
5. Counting the cell, and dilute the cell 107/ml.
[Question]
How to separate murine B and T lymphocytes?
23
Exp. VI Detection of T Lymphocytes Subgroup with the APAAP
[Principle]
T lymphocyte is one of the most functionally important cells in human immune
system. According to the CD (Cluster Differentiation) molecules expressed on its
surface, T cell is divided into two groups, CD3+CD4+CD8 － T cell and
CD3+CD4－CD8+T cell.
APAAP complex is consisted of alkaline phosphatase and mouse anti-alkaline
phosphatase antibody.
Samples (peripheral blood mononuclear cell, PBMC) are fixed on slides, followed
by the addition of primary antibody (mouse anti-human T cell CD3 McAb).
Bound antibody is detected by secondary/bridge antibody (rabbit anti-mouse IgG).
In this bridge antibody, one Fab is bound to primary Ab, the other Fab is bound to
APAAP complex. Afterwards, the substrate solution is added to the reaction
solution to demonstrate color reaction. Thus T cell subgroups expressing
corresponding CD molecules are stained with the pigment (Fig. 6-1).
Fig.6-l Principle of APAAP Method
[Application]
24
The APAAP method is employed to detect T cell subgroups in order to determine
the status of patient immune functions, to diagnose some related diseases. For
example, CD4/CD8 ratio is decreased in AIDS patients. It also used other cell
markers, such as MHC-I, MHC-II antigens.
[Materials]
1. The APAAP detection kit
2. Mouse anti-human CD3, CD4, CD8 McAb (primary Ab), rabbit anti-mouse IgG
Ab (secondary Ab)
3. PBS buffer
4. Mayer hematoxylin counterstain solution
5. 100% acetone
6. Centrifuge tube, test tube, pipette, centrifuge, microscope, slides and tips, etc
[Procedures]
1. Isolate PBMC by Ficoll-Hypaque density methods. Adjust PBMC
concentration to 1×106/ml with PBS buffer.
2. Take out 50-100μl cell suspension and add onto clean slide, air dry. Mark a
circle around the sample with crayon, fix 2 min with fixative 100% acetone.
Wash with PBS for 3 times.
3. Add 10μl primary Ab, incubate at 37℃ for 30 min in a moist container. Rinse
with PBS buffer for 5 min. Wipe the slide with paper towel. (* Student will
start from this step).
4. Add 10μl secondary Ab, 37℃, 30 min, rinse with PBS buffer, 5 min.
5. Add 10μl APAAP complex, 37℃, 30 min, rinse with PBS buffer, 5 min.
6. Add 20μl APAAP substrate onto slide, 37℃, 30 min, rinse with PBS buffer, 5
min.
7. Counterstain with Mayer hematoxylin solution for 1 min (this step is optional),
wash and stop reaction with running water from faucet.
8. Observe under 40 × microscope.
[Results]
Examine slides using 40 × microscope. Count 200 PBMC to determine
25
percentage of positive cells.
CD (+) cell: the cell nucleus stained blue, the cell membrane/plasma stained red.
CD (-) cell: the cell nucleus stained blue, no color or transparent on cell
membrane/plasma.
Reference Values:
CD3+ T cell: 60%-80%
CD4+ T cell: 35%-55%
CD8+ T cell: 20%-30%
CD4+ T/CD8+ T: 1.5-2.0
[Attentions]
1. Be sure to wipe liquid drops outside the circled cell area on the slide with
paper towel to prevent the added reagent in the circ1e from diffusing.
2. When adding Ab or other solutions onto the slide, the end of the pipette tips
can be used to spread the solution evenly on the slide.
3. When rinsing and washing with PBS solution or running water, be gentle so
that the cells won’t be washed away from the slide.
4. Glass slide should be pretreated with 1% BSA to make the cells easier to stick
to it.
[Questions]
1. What does the APAAP stand for?
2. In the above experiment, which reagent linked up the APAAP complex and the
anti-human CD McAb?
3. Based on the principle of enzyme immunoassay, please design an experiment to
determine the T cells subgroups.
26
Exp. VII
Measurement of Phagocytosis by Phagocytes
[Principle]
This protocol describes a simple assay to measure the ability of phagocytes to
bind and internalize or phagocytose bacteria (Fig.7-1). Bacteria and cells are
mixed in suspension and rotated to give optimal condition for interaction.
Extracellular bacteria are then removed by washing or centrifugation in sucrose
solution. The amount of phagocytosis is determined by examining the stained
cells under oil immersion microscope (Fig. 7-2).
Fig.7-l Mechanism of phagocytosis
Fig.7-2 Phagocyte
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[Application]
Determine the phagocytosis of macrophages and neutrophilic granulocytes and
evaluate the status of innate immunity of an individual.
[Materials]
1. Hank’s balanced salt solution (HBSS)
2. Monocytes/macrophages: e.g., cultured macrophage cell line, murine peritoneal
exudates macrophages or human PBMC
3. Overnight bacterial culture (e.g., candida albicans), live or heat-killed
4. Normal serum, fresh or freshly thawed and kept on ice
5. PBS containing 30% (w/v) sucrose (can be stored for months at 4℃ after filter
sterilizing)
6. PBS containing 5% (v/v) FCS (fetal calf serum)
7. Giemsa-Wright stain
8. Centrifuge tube, pipette, centrifuge, microscope, CO2 incubator, etc
[Procedures]
1. Wash monocyte/macrophage sample with 10ml of HBSS, centrifuge for 5 min
at l000r/min, 4℃ then discard supernatant. Repeat washing once and then
suspend cells in HBSS to a final concentration of 2.5×l07/ml.
2. Add 0.1ml macrophage suspension (2.5×106 cells) to 10mm×75 mm snap-top
tube. Prepare one tube for each condition (group) to be studied.
3. Vortex Candida albicans culture and dilute 1/10 in HBSS.
4. Vortex again, then transfer 0.1m1 of bacteria (2.5× 106 cells) to snap-top tube.
5. Add 50μl fresh or freshly thawed ice-cold normal serum. Add HBSS to lml.
Cap tightly and seal cap to tube with Parafilm.
6. Place tubes on a shaker and rotate end-over-end for 20 to 30 min at 8r/min,
37℃.
7. Two methods：
1 Basic method: Centrifuge tubes 8 min at 1000r/min 4℃. Remove supernatant,
○
add 2 vol ice-cold HBSS, and resuspend cells gently using a Pasteur pipette.
Repeat wash twice more to eliminate most residual extracellular bacteria.
Resuspend cells to desired concentration in ice-cold PBS/5 % FCS.
2 For more stringent removal of extracellular bacteria: Wash cells three times in
○
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HBSS as described in step 7a and resuspend in 1ml ice-cold HBSS. Using a
1ml plastic pipette, underlay with 1ml of 30% sucrose. Centrifuge 8 min at
1000r/min 4℃. Carefully remove HBSS and sucrose with a Pasteur pipette.
Resuspend pellet to desired cell density in ice-cold PBS/5 % FCS. (Cells are
usually suspended to 2ml so that cells are at a density of 106cells/ml for
centrifugation.)
8. Centrifuge 0.lml cells (l×105cells) onto a microscope slide by spinning 5 min at
650r/min, room temperature.
9. Stain slide with Giemsa-Wright mixture.
[Results]
Quantitate phagocytosis under oil-immersion microscopy (1000×), examining at
least 200 cells and counting the number of internalized bacteria in each one.
Calculate amount of phagocytosis according to the following formula: Phagocytic
index = (percentage of macrophages containing at least one bacterium) × (mean
number of bacteria per positive cell). This calculation takes into consideration not
only the number (percentage) of cells that are phagocytic, but also how
phagocytic they are — i.e., how many bacteria are internalized by each cell.
[Attentions]
1. Vortexing is important so that the bacteria are mixed to the single cells. For this
assay, a ratio of ten bacteria to one macrophage is used to allow easy
visualization and quantitation of phagocytosis. However, bacteria are wellphagocytosed and readily detectable at 1:1 ratio.
2. Time periods of the tubes on a shaker and rotate end-over-end >30 min should
not be used, because significant killing of many bacteria can occur quite rapidly
and dead bacteria degraded by extensive exposure to 37℃ may fail to be
stained and detected as phagocytosed.
3. The number of cells loaded into the cytocentrifuge may have to be varied with
cell size — if the macrophages are large, as some cell lines are, use fewer cells.
[Question]
In this experiment, what are the experimental factors which contribute to the
bacterial phagocytosis of macrophages?
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