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SSM Protocol
Collect blood.
Aliquot 100uLper tube.
Add different CD4 (verified clone) fluorochromes at standard concentration - 2ug/mL (0.5-2uL) - saturate the CD4s.
Stain 10 minutes RT.
Lyse / fix RBCs - 2mL (1x of BD RBC Lysing solution (CAT:349202)
Spin (300 RCF x 5min @ 4oC).
Decant
Add 4mL wash buffer
Spin (300 RCF x 5min @ 4oC).
Decant
Add 4mL wash buffer
Spin (300 RCF x 5min @ 4oC).
Decant
Resuspend in 300uL wash buffer.
*Make 4-5 tubes of unstained.
VOLTRATION (only negatives)
Let instrument warm up
Acquire unstained sample blood with voltages 0-1000 @ 25 or 50V increments for all detectors.
Gate in flowjo lymphocytes and export lymphocytes only.
Run negative lymphocyte script - (or manually determine rSD of negative lymphocytes and use voltage where rSD ~= 3-4x 0V).
Single Colour Tubes
Set all detectors to voltages determined.
Make a master sample mix - aliquot 5uL of single colour CD4 stains from all tubes (this is a time saver)
Acquire master sample and ensure all signals are on scale, if not on scale, stain at a lower concentration new CD4s to put positive on scale, or lower voltages.
If on scale acquire single colours - 3000-5000 lymphocytes per tube.
Calculate SSM in flowjo.