CD3／CD4／CD8Tcellsubsetcounts

Description
Procedure for CD3/ CD4 /CD8 T cell subset counts using CD3-FITC / CD4-PE / CD8-PECy5 antibody 

Procedure
1. One 5 ml round bottom tube (Falcon) is taken per blood sample to be analyzed. The tube is labeled, which includes the sample ID, test to be performed and date.
2. 15 ƒY´l of the Test antibody (CD3-FITC / CD8-PE / CD4-PECy5) (Cytognos) is added to each sample tube.
3. 100 ƒY´l of blood sample is added to corresponding test tube by REVERSE PIPETTING and the tubes are capped or covered with Parafilm. The tubes are mixed gently by tapping and incubated in dark for 10 min at room temperature. The tubes are shaken intermittently during incubation.
4. 2 ml of RBC lysing solution (1 x Quicklysis solution by Cytognos) is added to each tube. The tubes are then covered with Parafilm, mixed thoroughly and incubated in dark for 10 min at room temperature.
5. Sample tubes are then acquired on the Flowcytometer.
6. The tubes are analyzed immediately. 
7. If the samples are not going to be analyzed immediately, steps 1-4 are performed and the samples are stored at 40C in dark until further analysis. Step 5 is performed just before analysis.


Recipes
1. Round bottom tube (capacity 5 ml)
2. Test antibody (CD3-FITC/ CD8-PE/ CD4-PECy5; Cytognos, Spain)
3. Lysing solution (Quicklysis)