Methods in Immunology

1Immunology Methods forMedical Students

(Second Edition)

DepartmentDepartmentDepartment

Department

ofofof

of

ImmunologyImmunologyImmunology

Immunology

ChinaChinaChina

China

MedicalMedicalMedical

Medical

UniversityUniversityUniversity

University

SeptemberSeptemberSeptember

September

200720072007

2007

2PrefacePrefacePreface

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 PangWei.

Department of Immunology

Nov 11, 2006

3Exp. I Agglutination Reaction...1

i. Tube Agglutination Reaction.. 1

ii. SlideAgglutination 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 Assay16

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

4Exp. I Agglutination Reaction

Agglutination Reaction includes:1. Direct agglutination reaction: tube agglutination reaction and slide

agglutination reaction.2. Indirect agglutination reaction.

iii

i

...

.

TubeTubeTube

Tube

AgglutinationAgglutinationAgglutination

Agglutination

ReactionReactionReaction

Reaction

[Principle][Principle][Principle]

[Principle]

Agglutination is the interactions between insoluble particles (e.g. intact bacteriaand cells) or soluble antigens that attached to particles and their correspondingspecific antibodies that result in some visible agglutinates if given enough timeand the proper concentration of electrolyte.

Tube agglutination test is usually used in the semi-quantitative test, by mixing theknown particle antigen directly with serial dilutions of diagnostic serum in thetubes. 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 therelative agglutination amount.

[Application][Application][Application]

[Application]

A classic application of tube agglutination test method is the Widal test, adiagnostic procedure for detection of Salmonella infections, where the presenceof antibodies against Salmonella H and O antigens can be demonstrated in thepatients serum. It can help to diagnose Typhoid fever.

This experiment is to determine the antibody titer of rabbit anti-sheep-erythrocyteserum using sheep erythrocytes. The titer is the reciprocal of the last dilution of anantiserum capable of mediating some measurable effect such as precipitation oragglutination. It is a measure of the relative strength of an antiserum.

[Materials][Materials][Materials]

[Materials]

1. 1% fresh sheep erythrocyte;2. Rabbit anti-sheep erythrocyte serum (heat inactivation of complement at 56

for 30 min);

53. Saline, tube, pipette, test tube rack, etc.

[Procedures][Procedures][Procedures]

[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 transfer0.5ml of dilution to tube 3. Repeat the same procedure in tube 3,4,5,6 and 7 forserial dilution of serum and discard 0.5ml of dilution from tube 7. Notice: dontdo 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. Thefinal 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 (orovernight in incubator at 37).

Fig 1-1 Serial dilution of rabbit anti-sheep erythrocyte serum

[Resu[Resu[Resu

[Resu

lll

l

ts]ts]ts]

ts]

1. Take out the tubes from the water bath and do not shake the tubes whenobserving the results. First observe the control tube 8. There is no agglutinationin the control tube 8. It shows a red dot at the bottom of the tube result fromerythrocyte sedimentation. Then observe the results from tube 1 to tube 7. Theagglutination in the bottom of the tubes represents positive reaction. Theagglutination degrees are represented with (++++), (+++), (++) and (+)

6respectively.2. Assessment standards:1 (++++): represents all of the sheep erythrocytes agglutinated. A big piece ofagglutinant appears uniformly at the bottom of tubes and agglutinant brimcrimpled as petal, while the supernatant is clear.

2 (+++): represents about 75% sheep erythrocytes agglutinated. A piece ofagglutinant appears. The area of agglutinant is bigger than (++), while thesupernatant is a little turbid.

3 (++): represents about 50% sheep erythrocytes agglutinated. The area of theagglutinant becomes smaller and agglutinant brim becomes looser, while halfof the supernatant is turbid.

4 (+): represents about 25% sheep erythrocytes agglutinated. The erythrocytesedimentation at the bottom of the tube is surrounded by a little of agglutinant.

[Attentions][Attentions][Attentions]

[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 disturbingthe agglutinants during observation.

7iiiiii

ii

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.

SlideSlideSlide

Slide

AgglutinationAgglutinationAgglutination

Agglutination


Reaction


[Principle]

Slide agglutination reaction is the direct agglutination carried out on the slides, bydirectly mixing the antibody with a particle antigen under the certainconcentration of electrolyte. The result is positive when there is visibleagglutinate, otherwise negative.


[Application]

Slide agglutination is a qualitative test. It may be used mostly to determine bloodtypes (ABO types) or if antibody to bacteria is presented in blood as an indicationof infection of the bacteria. This experiment is to determineABO blood types.


[Materials]

1. Human peripheral blood2. Standard sera: Anti-A and anti-B sera3. Saline4. 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, 2and 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 slidefrom the box and mix the blood into the sera and saline on square 1,2 and3, respectively.

4. Shake slide gently, mix blood and sera well and observe the results in 1~2 min.

Fig 1-2. Blood type determination

1anti-A

2anti-B

3saline

8[Results][Results][Results]

[Results]

Positive result shows particle agglutination. Negative result appears uniformlyturbid. You can observe the results by microscope if the results are not clear.

[Results[Results[Results

[Results

Analysis]Analysis]Analysis]

Analysis]


[Attentions]

1. Slides require to be sterilized in 75% ethanol.2. Take blood with different sides of the brim of slide.

Bloodtype( phenotype)

Antigen on RBC Antibody in sera Gene type

A A Anti-BAb A/A,A/OB B Anti-AAb B/B, B/OO H Anti-AAb,Anti-BAb O/O(H)AB A, B ---- A/B

9iiiiiiiii

iii

...

.

IndirectIndirectIndirect

Indirect

AAA

A

gglutinationgglutinationgglutination

gglutination

III

I

nhibitionnhibitionnhibition

nhibition


Reaction

[Purpose][Purpose][Purpose]

[Purpose]

Pregnancy determination (Human Chorionic Gonadotropin, HCG detection inurine)


[Materials]

1. Urine samples ( with HCG)2. Standard HCG immunized latex granule3. StandardAnti-HCGAb4. Saline5. Black glass board

[Methods][Methods][Methods]

[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[Result[Result

[Result

observation]observation]observation]

observation]

Sandy agglutination in sample 2 and saline

[Conclusion][Conclusion][Conclusion]

[Conclusion]

Agglutination (+) HCG (--) Pregnancy (--)Agglutination (--) HCG (+) Pregnancy (+)

[Questions][Questions][Questions]

[Questions]

What are the applications of agglutination test?What is titer?How the antigen and antibody attach to each other?

10

Exp. II Precipitation Reaction

i. DoubleDoubleDouble

Double

ImmunodiffusionImmunodiffusionImmunodiffusion

Immunodiffusion


Reaction


[Principle]

Precipitation reaction occurs when soluble antigen (serum proteins, cell lysate ortissue lysate) binds its specific antibody and produce visible precipitate.

In Double Immunodiffusion Test, the solution of soluble antigen and antibody areadded into corresponding wells in the agar plate respectively, then let them diffusefreely. When the antigen meets with its corresponding antibody, they will combinewith each other. If the conditions (pH, temperature and concentration) areappropriate, there is a band of precipitate composed of the specific antigen and itsantibody. The characteristics of antigen and antibody can be analyzed according tothe existence of precipitation band, as well as its location and shape.


[Application]

Its mostly used to analyze the characteristics of antigen or antibody, determinethe purity of antigen and antibody or measure the titer of antibody. This test is todetect -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 (orumbilical cord blood)

3. Antibody: AFP diagnostic serum (anti-AFP antibody)4. Plastic plate (2.7cm7cm), agar-puncher, microsyringe, humid box, etc


[Procedures]

1. Preparation of agar plate: 3.5ml melted saline agar solution is poured into aplastic plate on the horizontal table. 7 wells with diameter of 3mm are punchedin the solidified agar with 5mm of interspace to each other. The pattern of wellslooks like a plum blossom.

11

2. Adding samples: sample serum, AFP positive serum and AFP diagnostic serumare respectively added into three wells by microsyringe, about 15l 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][Results][Results]

[Results]

The result is determined by the existence or nonexcistence of characteristic whiteband of precipitate. If an antibody is specific to the corresponding antigen, there isa complete fusion precipitate band between them. If two precipitate bands crosseach other when they meet, it indicates that two antigens are completely differentand 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 antibodiesand unknown antigen is partly identical with control antigens (Fig 2-2).

Fig 2-2


[Attentions]

12

1. When prepare the agar plate, pour the melted saline agar solution into a plasticplate rapidly, for the melted agar will solidify quickly.

2. Before punching the actual wells, practice at the corner of the agar plate.3. Dont lacerate the agar while punching wells and adding samples, in order toavoid any influence on the formation of the precipitate band.

[Question][Question][Question]

[Question]

What are the applications of the double immunodiffusion technique?

iiiiii

ii

...

.

SingleSingleSingle

Single

ImmunodiffusionImmunodiffusionImmunodiffusion

Immunodiffusion


Reaction

[[[

[

PrinciplePrinciplePrinciple

Principle

]]]

]

A melted agar solution mixed with specific antibody is poured into the plasticsplate. Then wells are punched and filled with known concentration of standardantigen which can diffuse into the agar and react with the antibody. When theantigen diffuses to a suitable concentration, it will react with the antibody andring-shaped bands of precipitate will be formed concentrically around the well.Its 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 theconcentration of the standard antigen. The standard curve can be obtained byusing the diameter as ordinate and the dilution rates as abscissa. The concentrationof the sample antigen can be calculated based on the diameter of its precipitationring according to the standard curve.

[[[

[

ApplicationApplicationApplication

Application

]]]

]

IgG, IgM, IgA, complement, transferrin, antitrypsin, glycoprotein, prealbumin andmany kinds of serum can be measured by this technique quantitatively. In thisexperiment, we will detect the concentration of human IgG in sample serum.

[[[

[

MaterialsMaterialsMaterials

Materials

]]]

]

1. Antigen: the tested human serum, the known concentration of IgG as referenceprotein (11.52mg/ml).

2. Antibody: anti-human IgG antibody mixed into 3.3% saline agar solution

13

4. Pipette, 2.7cm7cm plastic plate, puncher (D=3mm), microsyringe, humid box,and semi-logarithm coordinate paper.

[[[

[

ProceduresProceduresProcedures

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 isdetermined by the titer of antibody).

3 Mix agar with equal amount of diluted IgG antibody. Then pour solution intoplastic 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. Aftercomplete dissolving, the serum is diluted into a series of different dilutions. Forexample: the concentration of control serum IgG is 11.52mg/ml. Dilutions ofreference serum: 1:18, 1:36, 1:72, 1:144, 1:288. Concentration ofcorresponding IgG (g/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 afterits solidification according to figure 2-3. The distance between adjacent wells isabout 15mm.The agar is picked up from wells by needle. Be carefully about theedge of well.

4. Adding samples: Around 10-15l reference serum and sample serum is addedinto agar wells with different dilutions. Make sure the exact same amounts ofsamples 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.

[[[

[

ResultsResultsResults

Results

]]]

]

1. Take out agar plate, you will find milky white precipitate ring. Measure thediameter and write it down (Fig 2-3).

14

Fig 2-32. The reference curve: Measure the diameters of precipitin rings and draw astandard curve with dilution rates as ordinate and the diameters of rings asabscissa (Fig 2-4).

y = 2103.8e -0.3125x

050

100150200250300350

0 5 10 15 20Diameter

Dilution

Dilution

Expon. (Dilution)

Fig 2-4

3. Measure the IgG concentration of the sample serum: Measure the diameter ofthe precipitate ring of the sample and the concentration of sample IgG can becalculated from the equation.

[[[

[

AttentionsAttentionsAttentions

Attentions

]]]

]

1. This experiment belongs to quantitative test and could be influenced by manyeffect factor, for example, the quality of reference serum, the concentration ofantibody, the quality and concentration of agar, the thickness and uniform of theagar layer So it must be strictly controlled.

2. The reference curve can only be used once but not in other tests.

precipitate ringdiameters(mm)

15 13 11 9 6

Dilution rates 18 36 72 144 288

15

3. While mixing antibody into agar, the temperature of the agar solution shouldntbe too high in order avoid denaturalization and inactivation of the antibody.And it shouldnt be too low in order to avoid the agar solidify before spreadingout on the plate.

4. The samples must be add accurately and to avoid the alveolus formation in thewells.

5. The diameter must be checked accurately. If not, the error will be expanded forseveral times when multiplying by the dilution rates.

[[[

[

QuestionQuestionQuestion

Question

]]]

]

What are the principle and the application of single immunodiffusion technique?

16

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 rosettewith sheep erythrocytes by its E receptor. The number of E rosettes can imply Tlymphocytes number. This test is used to detect the number of T lymphocytesfrom peripheral blood.


[Application]

Separation of peripheral blood mononuclear cells (PBMC) and counting of Tlymphocytes in vitro.


[Materials]

1. Human peripheral blood2. Heparin solution, 500U/ml3. Lymphocyte separation medium(LSM), specific gravity 1.0770.001g/L4. 2% sheep erythrocyte5. Ca2+ and Mg2+ free Hanks balanced salt solution (HBSS)6. 0.5% methene blue staining solution7. Centrifuge tube, test tube, pipette, waterbath, centrifuge, microscope, etc.


[Procedures]

1. Preparation for lymphocyte suspension1 Draw 2ml human peripheral blood. Place into a test tube containing 0.1ml ofheparin and add equal volume of HBSS. Mix well. Pipe 1ml LSM into acentrifuge tube. Carefully overlay the LSM with 1ml diluted blood sample byslowly adding blood along the inner wall of test tube (Fig 3-1). The surface ofthe LSM must not be disturbed.

17

Fig 3-12 Centrifuge at 2000r/min for 20min at room temperature.3 Using a pipette, discard the plasma layer and harvest the mononuclear cell

band from tube at the interface between the plasma and LSM. Transfer themononuclear solution to a clean test tube containing 4ml HBSS (Fig 3-2).Mix well.

Fig 3-24 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. Thencentrifuge 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. Onedrop 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 rosetteforming cells from 200 viable lymphocytes. The lymphocyte is counted as a

18

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 around100:1.

3. Do not shake the test tube after E-rosette formation to prevent the rosettes fromfalling apart.

[Question[Question[Question

[Question

sss

s

]]]

]

What is the principle of E-rosette formation?How is the lymphocytes separated from the other ingredients of blood?

19

Exp. IV Enzyme-Linked Immunosorbent Assay (ELISA)


[Principle]

The principle of ELISA is based on the high specificity of antigen-antibodyreactions. It keeps immunogenic and activities after antigen or antibody absorbs tothe surface of solid-phase. ELISA may be quantified by determining with aspectrophotometer the rate at which the enzyme converts a clear substrate to acolored product. The assay is called enzyme-linked immunosorbent assay. ELISAis now widely used in many areas in the medicines.

Fig. 4-1 Principles of enzyme-linked immunosorbent assay (ELISA)

20

There are many different approaches to ELISA (Fig.4-1). They are typicallyperformed as a direct or an indirect method, but can also be performed as a twoantibodies sandwich method and a competitive assay. Here we introducesandwich ELISA.


[Application]

ELISA is a very sensitive laboratory method and can be quantitative when used inconjunction with standard curves. It can quantify the amount of antigen orantibody present in a given sample.

[Purpose][Purpose][Purpose]

[Purpose]

Double Abs sandwich ELISA detecting HBsAg in serum.

[[[

[

Materials]Materials]Materials]

Materials]

1. [Test Kits]1) HRP-labeled Ab 22) Positive control3) Negative control4) Concentrated eluant5) Chromogenic substrateA6) Chromogenic substrate B7) Termination solution8) Ab 1 coated wells9) Tape

2. Sample 1, 2, 33. Distilled water4. Micropipettor and tips5. Pipette6. Beaker7. Measuring cylinder8. Absorbent paper

[[[

[

ProceduresProceduresProcedures

Procedures

]]]

]

21

1. Add samples, 1 row of 12 wells for each 2 or 3 students. Pipet 50l of eachof these 5 elements--3 samples, the positive control and the negativecontrol--into 2 wells. The empty 2 wells are called blank. We will usedthese blank wells later for adjusting the microplate reader. Caution: Slowlydrawing fluid, you should empty the pipettor to the second stopping point,adding the samples slowly.

2. Pipet 50l 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 pipet2ml 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 overflowdont add too much eluant to any of the wells. Wait for 5-10 seconds beforediscarding the eluant from the wells. Repeat washing the wells 5 times.

5. Add 50l of chromogenic solution A into all 12 wells, and then addsolution B. Mix, tape, label and incubate at 37 for 15 minutes.

6. Terminate the reaction with 50l of termination solution in each well.7. Read the plate at 450nm for OD values

[Result[Result[Result

[Result

analysis]analysis]analysis]

analysis]

Cutoff value: COV= mean value of negative control OD values2.1Sample OD>=COV is positive, < COV is negativeLowest negative mean OD value is 0.05

[Attention][Attention][Attention]

[Attention]

1. Samples and test kit need to be used under room temperature2. Shake reagents before using3. Slowly draw and discharge samples with micropippettor4. Change tips between solutions5. Filter paper and tape cant be reused6. Wash wells carefully to prevent overflow7. Plate needs to be read within 10 minutes after adding the termination

solution[Question[Question[Question

[Question

]]]

]

What are the principles of ELISA?

22

Exp. V Separation of Murine T, B lymphocytes

iii

i

...

.

PreparationPreparationPreparation

Preparation

ofofof

of

murinemurinemurine

murine

splenolymphocytesplenolymphocytesplenolymphocyte

splenolymphocyte

sss

s


[Materials]

1. BALB/c mouse2. RPMI 1640, gentian violet3. Sterilized culture dishes, scissors, forceps4. Sterilized stainless steel screen (200-mesh)5. pipettes, tip, EP tube, syringe6. Centrifuge, light microscopes

[Procedure[Procedure[Procedure

[Procedure

sss

s

]]]

]

1. A mouse is sacrificed. Put the mouse into 70% alcohol for 10 minutes. Fix themouse lying on its abdomen on a board. Cleave its left back skin and exposethe 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 mixturewith 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 waterinto the tube and stand for 40s. Then add 1 ml 3.6% saline solution or resumeisotonic 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 culturemedium.

6. Counting the cell, and dilute the cell 107/ml.

iiiiii

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PreparationPreparationPreparation

Preparation

ofofof

of

murinemurinemurine

murine

thymocytethymocytethymocyte

thymocyte

sss

s


[Materials]

1. RPMI 16402. Cell culture flasks, pipettes, 96-well plates3. Centrifuge, light microscopes

23

[Procedure[Procedure[Procedure

[Procedure

sss

s

]]]

]

1. A mouse is sacrificed. Put the mouse into 70% alcohol for 10 minutes. Fix themouse lying on its abdomen on a board. Cleave its left back skin and exposethe 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 with2ml culture medium for 5min. Collect the suspension without dregs or filterthe mixture with gauze. The collected solution is centrifuged at 1,500 r/minfor 10 min.

3. The supernatant is discarded. Lyse RBCs by adding 3 ml sterile distilled waterinto the tube and stand for 40s. Then add 1 ml 3.6% saline solution or resumeisotonic 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 culturemedium.

5. Counting the cell, and dilute the cell 107/ml.


[Question]

How to separate murine B and T lymphocytes?

24

Exp. VI Detection of T Lymphocytes Subgroup with theAPAAP


[Principle]

T lymphocyte is one of the most functionally important cells in human immunesystem. According to the CD (Cluster Differentiation) molecules expressed on itssurface, T cell is divided into two groups, CD3+CD4+CD8 T cell andCD3+CD4CD8+T cell.

APAAP complex is consisted of alkaline phosphatase and mouse anti-alkalinephosphatase antibody.

Samples (peripheral blood mononuclear cell, PBMC) are fixed on slides, followedby 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 primaryAb, the other Fab is bound toAPAAP complex. Afterwards, the substrate solution is added to the reactionsolution to demonstrate color reaction. Thus T cell subgroups expressingcorresponding CD molecules are stained with the pigment (Fig. 6-1).

Fig.6-l Principle of APAAPMethod


[Application]

25

The APAAP method is employed to detect T cell subgroups in order to determinethe status of patient immune functions, to diagnose some related diseases. Forexample, CD4/CD8 ratio is decreased in AIDS patients. It also used other cellmarkers, such as MHC-I, MHC-II antigens.


[Materials]

1. TheAPAAP detection kit2. Mouse anti-human CD3, CD4, CD8 McAb (primaryAb), rabbit anti-mouse IgGAb (secondaryAb)

3. PBS buffer4. Mayer hematoxylin counterstain solution5. 100% acetone6. Centrifuge tube, test tube, pipette, centrifuge, microscope, slides and tips, etc


[Procedures]

1. Isolate PBMC by Ficoll-Hypaque density methods. Adjust PBMCconcentration to 1106/ml with PBS buffer.

2. Take out 50-100l cell suspension and add onto clean slide, air dry. Mark acircle around the sample with crayon, fix 2 min with fixative 100% acetone.Wash with PBS for 3 times.

3. Add 10l primary Ab, incubate at 37 for 30 min in a moist container. Rinsewith PBS buffer for 5 min. Wipe the slide with paper towel. (* Student willstart from this step).

4. Add 10l secondaryAb, 37, 30 min, rinse with PBS buffer, 5 min.5. Add 10l APAAP complex, 37, 30 min, rinse with PBS buffer, 5 min.6. Add 20l 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

26

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 cellmembrane/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 withpaper 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 tipscan be used to spread the solution evenly on the slide.

3. When rinsing and washing with PBS solution or running water, be gentle sothat the cells wont be washed away from the slide.

4. Glass slide should be pretreated with 1% BSA to make the cells easier to stickto it.

[Questions][Questions][Questions]

[Questions]

1. What does the APAAP stand for?2. In the above experiment, which reagent linked up the APAAP complex and theanti-human CD McAb?

3. Based on the principle of enzyme immunoassay, please design an experiment todetermine the T cells subgroups.

27

Exp. VII Measurement of Phagocytosis by Phagocytes


[Principle]

This protocol describes a simple assay to measure the ability of phagocytes tobind and internalize or phagocytose bacteria (Fig.7-1). Bacteria and cells aremixed in suspension and rotated to give optimal condition for interaction.Extracellular bacteria are then removed by washing or centrifugation in sucrosesolution. The amount of phagocytosis is determined by examining the stainedcells under oil immersion microscope (Fig. 7-2).

Fig.7-l Mechanism of phagocytosis

Fig.7-2 Phagocyte

28


[Application]

Determine the phagocytosis of macrophages and neutrophilic granulocytes andevaluate the status of innate immunity of an individual.


[Materials]

1. Hanks balanced salt solution (HBSS)2. Monocytes/macrophages: e.g., cultured macrophage cell line, murine peritonealexudates macrophages or human PBMC

3. Overnight bacterial culture (e.g., candida albicans), live or heat-killed4. Normal serum, fresh or freshly thawed and kept on ice5. PBS containing 30% (w/v) sucrose (can be stored for months at 4 after filtersterilizing)

6. PBS containing 5% (v/v) FCS (fetal calf serum)7. Giemsa-Wright stain8. Centrifuge tube, pipette, centrifuge, microscope, CO2 incubator, etc


[Procedures]

1. Wash monocyte/macrophage sample with 10ml of HBSS, centrifuge for 5 minat l000r/min, 4 then discard supernatant. Repeat washing once and thensuspend cells in HBSS to a final concentration of 2.5l07/ml.

2. Add 0.1ml macrophage suspension (2.5106 cells) to 10mm75 mm snap-toptube. 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 50l 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 methods1 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

29

HBSS as described in step 7a and resuspend in 1ml ice-cold HBSS. Using a1ml plastic pipette, underlay with 1ml of 30% sucrose. Centrifuge 8 min at1000r/min 4. Carefully remove HBSS and sucrose with a Pasteur pipette.Resuspend pellet to desired cell density in ice-cold PBS/5 % FCS. (Cells areusually suspended to 2ml so that cells are at a density of 106cells/ml forcentrifugation.)

8. Centrifuge 0.lml cells (l105cells) onto a microscope slide by spinning 5 min at650r/min, room temperature.

9. Stain slide with Giemsa-Wright mixture.


[Results]

Quantitate phagocytosis under oil-immersion microscopy (1000), examining atleast 200 cells and counting the number of internalized bacteria in each one.Calculate amount of phagocytosis according to the following formula: Phagocyticindex = (percentage of macrophages containing at least one bacterium) (meannumber of bacteria per positive cell). This calculation takes into consideration notonly the number (percentage) of cells that are phagocytic, but also howphagocytic 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 thisassay, a ratio of ten bacteria to one macrophage is used to allow easyvisualization and quantitation of phagocytosis. However, bacteria are well-phagocytosed and readily detectable at 1:1 ratio.

2. Time periods of the tubes on a shaker and rotate end-over-end >30 min shouldnot be used, because significant killing of many bacteria can occur quite rapidlyand dead bacteria degraded by extensive exposure to 37 may fail to bestained and detected as phagocytosed.

3. The number of cells loaded into the cytocentrifuge may have to be varied withcell 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 thebacterial phagocytosis of macrophages?

ii.Preparationofmurinethymocytes

Methods in Immunology

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