SIMONSIMMUNOFLUORESCENCE PROTOCOLS

 

 

DirectImmunofluorescence

 

 

Staining cells with antibodies directlylinked to fluorochromes is know as direct immunofluorescence (DIF). DIF lendsitself to multicolour experiments where a cell suspension is simultaneouslystained with two, three or four antibodies each tagged with a differentfluorescent dye. When designing multicolour experiments it is important to usedyes that are compatible with each other, if in doubt speak to Simon. Theinclusion of Sodium Azide prevents "capping" and so enables shorter incubationsat room temperature to be used. If you are in any doubt about whichfluorochromes can be used on our instrument (FACS Calibur) or any other aspectof your experiment design then come and see Simon ph 5188.

 

Controls

 

All multicolour experiments requirecompensation controls where the same cells are stained with each of thefluorochromes separately. The correct negative control for allimmunofluorescent experiments are cells treated in the same way but incubatedwith isotype matched control antibodies with no known specificity tagged withthe same fluorochromes as the test antibodies. Sometimes when dealing withsuspensions where there will be negative and positive cells such as peripheralT-Cells it is permissible to consider the unstained cells in the sample thenegatives and exclude the negative control.

 

Equipment and Reagents

 

Tissue culture medium with 10mM SodiumAzide (TCM-N3)

Wash Buffer: DPBS with 10mM Sodium Azide(DPBS-N3)

Fixative :1%paraformaldehyde in PBS (PFA) (see notes 1)

Bench centrifuge

12X75mm plastic test tubes

Antibodies

 

Method

 

1. Harvest cellsin usual way, do not use trypsin to detach adherent cells, rinse cells withcation free PBS then use EDTA in PBS (see notes 2). Wash cells in TCM-N3 by centrifuging in 50ml conical tubesfor 10 minutes @2000rpm. Aspirate supernatant and resuspend in 1ml (TCM-N3). Count your cells using ahemocytometer. The ideal number of cells is about 5x105 per tube and less than 5x104is too few. Resuspend in nXml of TCM-N3where n=the number of tubes you will have.

 

2. Pipette 1ml ofthe cell suspension into each of your 12x75mm tubes (having labelled themfirst). Centrifuge for 5 minutes @1500rpm. Aspirate all but about 10µl of thesupernatant. Resuspend cells by vortexing or flicking the tube.

 

3. Dispense your antibodies intomicrocentrifuge tubes, with multicolour experiments all the antibodies for onetube can be pre-mixed before addition to cells. Centrifuging briefly in amicrocentrifuge removes aggregates and reduces background staining. Add theappropriate amount of antibody to each tube (read manufacturers suggestions)and incubate for 15 minutes at room temperature.

 

4. Resuspendcells by flicking the tube or vortexing gently and add 1ml of DPBS-N3,centrifuge tubes at 1500rpm for 5 minutes. Aspirate supernatant, resuspendcells by flicking or vortexing. Add 0.5ml of 1% formaldehyde in PBS by addingslowly while vortexing gently. Samples should be left for at least one hour inthe formaldehyde solution before running on cytometer if they are virusinfected or possibly virus infected.

 

Notes 1

 

Paraformaldehyde is the solid form ofpolymerized formaldehyde. Formaldehyde solution should be made fresh and notkept for more than a week. The most convenient form is to buy vials of 20%formaldehyde from Tousimis cat#1008A. Old formaldehyde can spoil you data byincreasing non specific background fluorescence. If you have to make it up fromthe powdered paraformaldehyde, mix the powder with PBS and leave in a waterbathfor a day or two. Heating on a hotplate is not recommended as the fumes aredangerous.

Notes 2

 

One method of removing adherent cellsfrom flasks is as follows:

Make up PBS with 0.53mM EDTA, it isimportant that there are no divalent cations i.e. Ca2+ or Mg2+ in the PBS.

Wash the monolayer free of tissue culturemedium with PBS-EDTA, decant off fluid and add more PBS-EDTA, incubate for 15minutes at 35°C, rocking the flask may help. For really stubborn cells you may need to give the flask a fewhard raps to detach them.

 

IndirectImmunofluorescence

 

 

 

Staining cells with antibodies that arenot directly conjugated to fluorochromes then using a second labelled reagentto bind to your primary antibody is know as indirect immunofluorescence (IIF).This method was pretty standard before so many antibodies became availabledirectly conjugated to fluorochromes. Mostly with unlabelled antibodies thesecond step reagent will be another antibody with a specificity for the primaryantibody allotype or sometimes isotype. Typically antibodies against humanantigens and made in mice so are mouse immunoglobulins usually mouse IgG1, thetypical second step reagent would be goat anti-mouse IgG conjugated to FITC.However care should be taken to ensure your second step reagent will recognizethe primary antibody for instance if your primary antibody is mouse IgG2a andyour second step reagent is anti-mouse IgG1 then it will not work. Also someprimary antibodies are made in hamster or rat, beware, sorry to labor thispoint.

 

Some primary antibodies are soldconjugated to biotin, in this case the second step reagent will be afluorochrome labelled streptavidin or avidin. The affinity of streptavidin forbiotin is awesome so short incubations can be used.

 

Multicolour experiments are much morediffecult with indirect immunofluorescence, although possible. However thefollowing protocol assumes its a single colour experiment. If you are in anydoubt about which fluorochromes can be used on our instrument (FACS Calibur) orany other aspect of your experiment design then come and see Simon ph 5188.

 

Controls

 

The correct negative control for allimmunofluorescent experiments are cells treated in the same way but incubatedwith isotype matched control antibodies with no known specificity tagged withthe same fluorochromes as the test antibodies. Sometimes when dealing withsuspensions where there will be negative and positive cells such as peripheralT-cells it is permissible to consider the unstained cells in the sample thenegatives and exclude the negative control.

 

Equipment and Reagents

 

Tissue culture medium with 10mM SodiumAzide (TCM-N3)

Wash Buffer: DPBS with 10mM Sodium Azide(DPBS-N3)

Fixative :1%paraformaldehyde in PBS (PFA) (see notes 1)

Bench centrifuge

12X75mm plastic test tubes

Antibodies

 

 

Method

 

1. Harvest cellsin usual way, do not use trypsin to detach adherent cells, rinse cells withcation free PBS then use EDTA in PBS (see notes 2). Wash cells in TCM-N3 by centrifuging in 50ml conical tubesfor 10 minutes @2000rpm. Aspirate supernatant and resuspend in 1ml (TCM-N3). Count your cells using ahemocytometer. The ideal number of cells is about 5x105 per tube and less than 5x104is too few. Resuspend in nXml of TCM-N3where n=the number of tubes you will have.

 

2. Pipette 1ml ofthe cell suspension into each of your 12x75mm tubes (having labelled themfirst). Centrifuge for 5 minutes @1500rpm. Aspirate all but about 10µl of thesupernatant. Resuspend cells by vortexing or flicking the tube.

 

3. Dispense your antibodies intomicrocentrifuge tubes, Centrifuging briefly in a microcentrifuge removesaggregates and reduces background staining. Add the appropriate amount ofantibody to each tube (read manufacturers suggestions) and incubate for 15minutes at room temperature.

 

4. Resuspendcells by flicking the tube or vortexing gently and add 1ml of DPBS-N3,centrifuge tubes at 1500rpm for 5 minutes. Aspirate supernatant.

 

5. Repeat step 4.leaving about 10µl of fluid in the tube, add the second step reagent, incubatefor 15 minutes at room temperature. (incubations can probably be reduced withstreptavidin.

 

6. Resuspendcells by flicking the tube or vortexing gently and add 1ml of DPBS-N3,centrifuge tubes at 1500rpm for 5 minutes. Aspirate supernatant, resuspendcells by flicking or vortexing. Add 0.5ml of 1% formaldehyde in PBS by addingslowly while vortexing gently. Samples should be left for at least one hour inthe formaldehyde solution before running on cytometer if they are virusinfected or possibly virus infected.

 

 

 

Notes 1

 

Paraformaldehyde is the solid formpolymerized formaldehyde. Formaldehyde solution should be made fresh and notkept for more than a week. The most convenient form is to buy vials of 20%formaldehyde from Tousimis cat#1008A. Old formaldehyde can spoil you data byincreasing non specific background fluorescence. If you have to make it up fromthe powdered paraformaldehyde, mix the powder with PBS and leave in a waterbathfor a day or two. Heating on a hotplate is not recommended as the fumes aredangerous.

 

Notes 2

 

One method of removing adherent cellsfrom flasks is as follows:

Make up PBS with 0.53mM EDTA, it isimportant that there are no divalent cations i.e. Ca2+ or Mg2+ in the PBS.

Wash the monolayer free of tissue culturemedium with PBS-EDTA, decant off fluid and add more PBS-EDTA, incubate for 15minutes at 35°C, rocking the flask may help. For really stubborn cells you may need to give the flask a fewhard raps to detach them.

 

 

 

 

 

 

 

IMMUNOFLUORESCENCEEXTRAS

 

 

Blocking Fc ReceptorsDuring Immunofluorescence Staining

 

Some cell types have a lot of Fcreceptors the cross react with mouse Igs to produce a lot of non specificstaining. Monocytes, macrophages and B-cells in particular can cause problems.Saturating these receptors with human immunoglobulin before staining withantibodies usually greatly improves results.

 

1. Prepare cells in the usual way, spin them down andaspirate supernatant.

 

2. Resuspend cell pellet in 50µl 10% heat inactivatedhuman serum in PBS. Incubate for 15 minutes at room temperature.

 

3. Without washing add your antibody and incubate foryour usual time.

 

4. When washing cells use the same 2% human serum PBS.If you are going to fix the cells the last wash should be just PBS (straightup!).

 

Dual ImmunofluorescenceStaining When One Antibody Is Directly Conjugated And The Other Is Not

 

1. Prepare cells in the usual way, spin them down andaspirate supernatant.

 

2. Incubate cells first with the unconjugatedantibody, wash then incubate with the second step reagent (usually goat antimouse-fitc or PE) and wash again (twice).

 

3. Incubate cells with 20µl 10% mouse serum (heatinactivated) in PBS for 10 minutes at room temperature.

 

4. Add without washing your directly conjugatedantibody, incubate as usual, wash and fix.

 

ImmunofluorescenceStaining Of Lymphocytes Using Whole Blood Method

 

 

1. Draw bloodinto tubes containing EDTA. Immediately before staining cells invert the tubehalf a dozen times to suspend cells.

 

2. Dispense100µl of blood into Falcon 2054 tubes. Add the recommended amount of antibody(usually 20µl with BD antibodies). (If you are not looking for very rare cells50µl blood and 10µl AB can be used). Incubate for 10 minutes at roomtemperature in the dark. When staining with more than one antibody if theantibodies are directly conjugated they can all be added at the same time.

 

 

3. Add1ml of 1X FACSlyse (0.5ml if you halved the volumes, incubate 10 minutes atroom temperature. Centrifuge @1500rpm for 5 minutes

 

 

4. Resuspendpellet by flicking the tube, add 1ml DPBS with 10mM sodium azide, centrifuge@1500rpm for 5 minutes.

 

5. Repeatstep 4

 

6. Resuspendpellet by flicking the tube, add 0.5ml 1% paraformaldehyde in DPBS slowly whilegently vortexing the cells.