NB. mcl = microlitre and degC = degrees centigrade
1) Aliquot 25mcl of each of the test antibodies at the required dilution (neat tissue culture sups. or 10mcg/ml) into wells of a 96 well microtitre plate, remembering to include positive and negative (irrelvant isotype, no antibody) controls.
2) Prepare single cell suspension of cells to be labelled (eg. peripheral blood mononuclear cells from "LymphoPrep", or total white cells by water lysis of whole blood), well washed, and resuspended into "labelling buffer" (eg. phosphate buffered saline [PBS] containing 0.5% BSA plus 0.1% NaN3, and 5% heat inactivated [56degC, 45min] normal rabbit serum [HINRS] to block Fc and non-specific Ig binding sites).
3) Add 25mcl of cell suspension containing approx. 1 million cells to each antibody well, mix on plate shaker, and incubate at 4degC for 1 hour.
4) Wash cells by filling wells to 200mcl with "wash buffer" (eg. PBS + 0.1% BSA + 0.1% NaN3) and pelletting cells in a centrifuge with plate carriers at 1000rpm. for 2 mins. Suck off liquid (or flick if experienced!) and resuspend pellet on a plate shaker. Wash twice more as above.
5) Add 25mcl detecting antiglobulin fluorescent conjugate directly to the cell pellets at appropriate dilution (as we often are using both rat and mouse monoclonal antibodies we might use a mix of FITC-goat anti-mouse Ig plus FITC-goat anti-rat Ig - eg. Sigma Cat Nos. F0257 and F6258 mixed together each at 1/100) in "labelling buffer". This should also contain 5% heat inactivated serum (same species as the target cells) to block cross reaction with surface Ig whenever cells have been in recent contact with serum (eg fresh tissues) or if the sample is likely to contain any cells with expressed or aquired surface Ig (eg. B cells or neutrophils). Mix well on plate shaker and then incubate at 4degC for 1 hour.
6) Wash as in step 4 above a total of three times.
7) Resuspend cell pellets thoroughly in 75mcl PBS containing 1% BSA + 0.1% NaN3, then add an equal volume of 4% Formalin in PBS to fix cells for later analysis by flow cytometry. These fixed and labelled cells can then be stored before analysis by sealing the plate and storing in the dark at 4degC for many weeks if necessary.
8) On analysis, scatter gating should be used to avoid collecting data from debris and any dead cells - also make sure the cells are resuspended well first. Alternatively, labelled cells can be analysed without fixing, but only immediately, and this allows the use of vital stains such as propidium iodide (eg. at 10mcg/ml) to exclude dead cells (if you do this please use the FACSort and change the sheath from water to PBS).
9) If you have a choice of LINEAR or LOGARITHMIC amplifiers for the fluorescence signal, it is generally preferable to choose the latter as this minimizes the effects of different sensitivities between machines for this type of data collection.
Dual colour method using local biotin or PE conjugate (after indirect detection of first antibody)
1) Proceed to label cells by single colour indirect method steps 1 through 6. Be sure to include additional controls where either no first antibody or second mAb conjugate are added to allow you to check at the analysis stage for correct colour compensation (I am assuming that 2 colour analysis with more "normal" protocols (ie. direct conjugates) is routine for you if you are going to attempt this).
2) To the single colour labelled cell pellets add 25mcl of the second antibody in a form that can be used in dual colour analysis, at an appropriate dilution in PBS + 1% BSA + 0.1% NaN3 + 2% of a mix of normal mouse plus rat serum (essential to inhibit secondary capture by the first antiglobulin). This could be either a mAb that has been directly conjugated with R-Phycoerythrin (PE) or alternatively a direct mAb conjugate with biotin to be followed later by Streptavidin-PE (see below). You should not attempt to use a further antiglobulin conjugate with this method. As an example, we might use anti-CD3 conjugated to biotin to identify T-cells by two colour analysis.
3) Mix well on plate shaker and incubate at 4degC for 1 hour.
4) Wash a total of three times as above (see step 4 of single colour method).
5) If you used a direct PE conjugate above then resuspend and fix for analysis (see step 7 of single colour method) immediately, and analyse as in step 8 below. Otherwise proceed to step 6 below.
6) If your second antibody was a biotin conjugate then add 50mcl Streptavidin-Phycoerythrin (SA-PE) conjugate diluted in PBS + 0.1% BSA + 0.1% NaN3 - we use SA-PE Cat. No. STAR 4B from Serotec, Oxford, UK., at a dilution of 1/200. Incubate at 4degC for 1 hour, then wash once in biotin containing medium (eg IMDM: this helps avoid clumping and quenching by the multivalent streptavidin congugate) + 0.1% BSA + 0.1% NaN3, followed by three washes as in step 4 above.
7) Resuspend and fix for analysis as in step 7 of single colour method.
8) For analysis it is often simplest to gate on both the scatters and the second (PE) colour, to generate data from fluorescence (FITC) histograms of the population identified by your second mAb conjugate (eg. CD3 positive T-cells in the example of step 2 above). In this manner you can generate data on more specific subsets of, for example, T cells in peripheral blood.