Protocol: Direct Immunofluorescence Staining of Immunoglobulin Light Chains on B Lymphocytes in Whole Blood

For use with AbD Serotec’s directly-conjugated dual colour reagents recognising human kappa and lambda immunoglobulin light chains.

The immunofluorescent staining of immunoglobulin expression by B lymphocytes in whole blood requires a procedure to remove serum immunoglobulins (which otherwise interfere and block staining with immunoglobulin-specific antibodies).

Note: Specific methodology for blood appears in [] brackets

Reagents:

PBS/BSA

Phosphate Buffered Saline pH 7.4 20mM containing glucose and 1% Bovine Serum Albumin

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

1. Collect blood into appropriate anti-coagulant - AbD Serotec recommends the use of EDTA, although satisfactory results may also be obtained using heparin or acid-citrate dextrose.
2. Aliquot 2-3 ml of whole blood into a 25 ml universal container. Then add 20-25 ml of PBS/BSA, pre-warmed to 37°C and mix well.
3. Centrifuge at 400 g for 5 minutes. Carefully aspirate the supernatant taking care not to disturb the cell pellet, and resuspend the pellet in the residual supernatant.
4. Repeat steps 3 and 4 twice more (three washes in total).
5. Aliquot 100 ul of the washed blood into the required number of test-tubes. Add appropriate volume of AbD Serotec antibody at the recommended dilution (see specific datasheet). Mix well and incubate at room temperature for 30 minutes.
6. Add 2 ml of freshly prepared AbD Serotec Erythrolyse (BUF04 and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes. Discard supernatant.
7. Wash with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the supernatant.
8. Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS/BSA if required.
9. Acquire data by Flow Cytometry.