|
For use with 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: This method provides a general
procedure for use with the majority of 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 - Serotec recommends the use of EDTA, although satisfactory
results may also be obtained using heparin or acid-citrate
dextrose. 2 Aliquot 2-3ml of whole blood into a 25ml
universal container. Then add 20-25ml of PGB, pre-warmed to
37oC and mix well. 3. Centrifuge at 400g 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 100ml of the washed blood into the
required number of test-tubes. Add appropriate volume of Serotec antibody
at recommended dilution (see specific datasheet for details). Mix well and
incubate at room temperature for 30 minutes. 6. Add 2ml of freshly prepared Serotec
Erythrolyse (Cat. No. BUF04) and mix well. Incubate for 10 minutes at room
temperature. Centrifuge at 400g for 5 minutes. Discard
supernatant. 7. Wash with 2ml of PGB, centrifuge at 400g for
5 minutes. Discard supernatant. 8. Resuspend cells in 0.2ml of PGB or with
0.2ml of 0.5% paraformaldehyde in PBS if required. 9. Acquire data by Flow Cytometry. Notes: Appropriate control samples should always be
included. We recommend the inclusion of an isotype-matched control in each
case - please contact Serotec's Technical Services department for details
of available reagents. The cell washing procedure described above has
no effect upon other cell surface antigens, and has been shown not to
affect the recovery of individual cell subsets. Solutions used: PGB =
Phosphate Buffered Saline pH 7.2-7.4 containing 20mM glucose and 1% Bovine
Serum Albumin Reference: Reynolds, W.M. et al. (1992) A simple
technique for the determination of k and l immunoglobulin light chain
expression by B cells in whole blood. J. Imm. Meths.
151:123-129.
|