PBMCs were stimulated with anti-CD3 mAb (OKT3) plus CD28, or mixed in a 1:1 ratio with irradiated allogeneic DCs in mixed lymphocyte reaction (MLR) as described (18 (link)). To determine cell proliferation, T cells were labeled with CFSE and cell division was monitored by using the FITC channel in a FACScanto II flow cytometer (BD Biosciences). Aliquots of PBMCs were analyzed by 7-color flow cytometry using a panel of surface molecule specific antibodies: CD4-APC (5 ug/ml), CD8-Pacific Blue (5 ug/ml), CD62L-APC/Cy7 (5 ug/ml), CD45RA-PE/Cy5 (2.5 ug/ml) and CD45RO-PE/Cy7 (2.5 ug/ml) (BD Pharmingen, San Jose, CA). Analyses of CD8+ T cell subsets were determined by assessing CD45RA and CD62L surface markers.
Protocol detail
Monitoring T Cell Proliferation and Subset Analysis
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Cd45ro
Cd62l
Cd8 t cell
Cell division
Cell proliferation
Cfse
Fitc
Flow cytometry
Mixed lymphocyte reaction
Okt3
Surface antibodies
T cells
Corresponding Organization :
Other organizations : The University of Texas MD Anderson Cancer Center, Cape Town HVTN Immunology Laboratory / Hutchinson Centre Research Institute of South Africa
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Variable analysis
independent variables
- Anti-CD3 mAb (OKT3) plus CD28 stimulation
- Mixed lymphocyte reaction (MLR) with irradiated allogeneic DCs
- T cell proliferation monitored by CFSE labeling and cell division using FITC channel in flow cytometry
- CD8+ T cell subset analysis by assessing CD45RA and CD62L surface markers
- PBMCs were analyzed by 7-color flow cytometry using a panel of surface molecule specific antibodies: CD4-APC (5 ug/ml), CD8-Pacific Blue (5 ug/ml), CD62L-APC/Cy7 (5 ug/ml), CD45RA-PE/Cy5 (2.5 ug/ml) and CD45RO-PE/Cy7 (2.5 ug/ml)
- Positive control: PBMCs stimulated with anti-CD3 mAb (OKT3) plus CD28
- Negative control: Not explicitly mentioned
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