Immunofluorescent staining of lymphocyte membranes

8.2.1 Immunofluorescent staining of lymphocyte membranes

This method is generally applicable to antisera of any specificity used with any viable cell suspen- sion. Antisera dilution used in this experiment should be optimized. Since there is individual variation in antibody affinity and avidity, a wide range of antibody concentrations should be used in the initial standard experiment to ensure reasonable fluorescence. (Obviously cost will, to

a certain extent, limit the range used.)

Immunofluorescent staining

MATERIALS AND EQUIPMENT Lymphoid organs, e.g. lymph node, spleen Tissue culture medium containing 0.1% w/v bovine serum albumin (BSA) and 20 m M sodium azide Nylon wool 2-ml syringe barrels Haemocytometer Rabbit anti-mouse immunoglobulin (anti-Ig) Fluorescein-conjugated goat, pig or sheep anti-rabbit immunoglobulin (FITC anti-Ig) Mounting medium: 70% glycerol, 30% glycine–saline buffer, pH 8.6

Note: Azide is a dangerous chemicalado not discard down the sink.

8.2LYMPHOCYTE SURFACE MEMBRANE

METHOD

1 Prepare a lymphocyte suspension from blood or one of the solid lymphoid organs.

2 Filter cell suspensions from lymphoid organs through nylon wool to remove aggregates.

3 Wash all cells three times in tissue culture medium by centrifugation (150 g for 10 min at 4°C).

4 Resuspend the cells in 10 ml of medium and count the number of lymphocytes per ml using

a haemocytometer and phase-contrast microscope.

5 Pipette out 2 aliquots of approximately 10 7 lymphocytes of each cell suspension and centrifuge to obtain a pellet (150 g for 10 min at 4°C).

6 Add 0.1 ml of the required dilution of rabbit anti-mouse Ig or normal rabbit serum (NRS) to

1 aliquot of each cell type.

7 Incubate for 30 min on ice.

8 Wash twice by centrifugation (150 g for 10 min at 4°C) to remove unbound protein.

9 Add 0.1 ml of the fluorescein-conjugated anti-rabbit Ig (1 mg/ml total protein) to all cell pellets.

10 Incubate for 30 min on ice.

11 Wash three times by centrifugation (150 g for 10 min at 4°C) to remove the unbound conjugate.

12 Resuspend, add 1 drop of glycerol–glycine mounting medium to the dry cell pellet and mix thoroughly.

13 Put 1 small drop of cell suspension on a microscope slide, add a coverslip and ring with nail varnish.

14 Examine the cell preparations under an incident light UV microscope and identify

lymphocytes visible as green rings. This is characteristic of cell-surface staining of viable lymphocytes. In addition, note the homogeneously stained dead cells that will inevitably be present.

15 For each microscope field, count the number of fluorescently stained lymphocytes (green) and then the total viable lymphocytes viewed under phase contrast.

16 Count a total of 200 cells under visible light and calculate the percentage of fluorescing (positive) lymphocytes for each preparation.

TECHNICAL NOTES • Staining viable cells confines the antibody to the external surface membrane. Staining of

internal components can only be accomplished efficiently by cell fixation and membrane permeabilization.

• If required, the method may be abbreviated by exposing the cells to each antibody for 10 min on ice, with only slight loss of sensitivity. • Precisely the same method may be used to prepare cells for cytofluorimetric analysis. • Several commercial companies now market an antifade fluorescent mounting medium. It is

particularly advantageous to use an antifade compound for photomicroscopy, and also to remember that the quenched fluorochrome slowly recovers. A cheaper but slightly less effective antifade mountant may be prepared as described below.

C H A P T E R 8: Lymphocyte structure

Antifade mountant

MATERIALS p-phenylenediamine Phosphate-buffered saline (PBS) Glycerol

0.5 M carbonate–bicarbonate buffer, pH 9.0

METHOD

1 Dissolve 100 mg p-phenylenediamine in 10 ml of PBS in the dark.

2 Add 90 ml of glycerol and mix thoroughly.

3 Adjust to pH 8.0 with the carbonate–bicarbonate buffer.

4 Store the mixture at –20°C in the dark.

5 Use as glycerol–glycine mountant, step 12 in Method above. Prepare fresh antifade mountant when the stock solution turns brown.