The antibodies were obtained from BD Biosciences (San Jose, CA) and included: HLA/DR/DP/DQ FITC: catalog no.: 555558, HLA-A,B,C Phycoerythrin: catalog no.: 555553, CD10 (APC) BD catalog no.: 340923, CD19 (APC-H7) clone: SJ25C1 BD catalog no.: 560177, CD34 (Per CP-Cy5.5) BD catalog no.: 347203, and CD45 (V450) clone: H130 BD catalog no.: 560367.
After 6 7 days, proliferating CD4+ (CD3+CD8−) and CD8+ (CD3+CD8+) T cells were determined by direct staining with mAbs conjugated with α-CD3-Per-CP and α-CD8-PE.
Cells were washed 3 times with chilled PBS and then incubated with secondary antibody conjugated with R-phycoerythin or Peridinin chlorophyll protein (R-PE or Per-CP, 1 µg) in 50 µl of PBS with 10% FBS for a further 30 minutes at 4°C in the dark.
For CXCR4 detection, 10 μl of PE-conjugated anti CXCR4 (BD, clone 12g5) was used in combination with anti CD45 Per-CP and CD19 Pe-Cy7.
The antibodies employed were CD117-APC/Alexa Fluor750 (immature myeloid cells); CD34-FITC (hematopoietic stem cells); CD45-Per-CP (pan leukocyte marker) and Gr-1-APC/Alexa Fluor750 (immature myeloid cells) (Biosciences Pharmingen, San Diego, CA, USA).
The following directly conjugated monoclonal antibodies were used: anti-CD3-Pacific Blue, anti-CD4-AmCyan, anti-CD25-Per-CP-Cy5·5, anti-CD161-PE, anti-CD39-APC, anti-CD147-FITC (BD Bioscience) and anti-CD127 PE-Cy7 (eBioscience).
Cells were first fixed and permeabilised with Triton X-100 0.02%, then stained with fluorescein isothiocyanate (FITC -conjugated, allophycocyanin (APC), peridin chlorophyll protein (Per-CP) and phycoerythrin-conjugated monoclonal antibodies (mAb) and their respective isotype-matched controls (Southern Biotechnology Associates, Birmingham, AL, USA).
After stimulation of CD3+CD8+ and NK cells, washing of the cells in PBS and resuspension in paraformaldehyde, receptors can be measured as follows: CD3-Per-CP; CD8-APC-H7; CD38-PE-Cy7; CD107b-FITC CD107b-FITC; CD16-PE; CD56-PE (BD Biosciences, Australia).
Cells were washed with PBS 1% bovine serum albumin (BSA) and incubated with anti-CD4-FITC and anti-CD8-Per-CP antibodies (eBioscience) for 30 minutes and then washed twice with PBS 1% BSA followed by detection of incorporated EdU in accordance with the manufacturer's instructions.
After culture, 100 μL of sample will be stained with the following conjugated fluorochromes for 15 min at room temperature (CD3-FITC; CD4-Per-CP; CD25-APC; CD134-PE, BD Biosciences, Australia), followed by washing in PBS (1 mL), resuspension in 0.5 mL of 0.5% paraformaldehyde (Sigma-Aldrich, Australia) and then multi-parameter flow cytometric analysis.
