OBJECTIVE
The same as previous ones. In this case changing the CD3 color (BD, CD3-PE_Cy7 clone SK7)
MATERIALS
Same as previous experiments, except the CD3.
PROCEDURE
- Isolate PBMCs from healthy donor with Ficoll-Paque (followed Ficoll protocol)
- Washed 2x with PBS and re-suspend PBMCs in PBS-BSA 2%. Count them and dilute to final concentration of 1mill/mL
- Take 1mill cells (1mL) per condition and aliquote to FACS tubes.
- Spin again and re-suspend in PBS/BSA (100uL)
- Add antibodies in 8 different tubes (1uL of each antibody):
Tube 1: no antibodies, just cells
Tube 2: CD19-FITC
Tube 3: CD3-PE_Cy7
Tube 4: CD27-PerCP-Cy5.5
Tube 5: CD38-PE
Tube 6: CD138-PE
Tube 7: CD3-PECF-594 + CD19-FITC + CD27-PerCP-Cy5.5 + CD38-PE
Tube 8: CD3-PECF-594 + CD19-FITC + CD27-PerCP-Cy5.5 + CD138-PE
- Incubate 45′ in ice
- Add 1mL of cold PBS/BSA 2%. Spin down at 1000g 5′ 4ºC
- Re-suspend in 1mL of PBS/BSA and spin again.
- Re-suspend in 2mL of PFA 4% in PBS
- Analyze
RESULTS
This time it worked pretty well. I think is the way to proceed. I have to improve cd19 signal, but otherwise compensation is pretty good. The percentages of CD3- CD19+ CD27+ CD38+ are 0.06% of total and the percentage of CD3- CD19- CD27+ CD138+ are 0.17%
CONCLUSION
Ill stick with these antibodies. Populations, at least in controls, are pretty unfrequent.
