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If you have any questions, please refer to the University's data classification guide or contact ict.askcyber@sydney.edu.au

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.