Antibody combination panels for four or five color immune-staining were as follows:

CD3PerCP, CD4 FITC, CD25APC, and rat Foxp3PE (all from eBioscience, San Diego, CA).

CD4 PercpCy5.5, HLA-DR APC, CD127 Pac Blue and rat Foxp3 PE (eBioscience, San Diego, CA).

CD4 PercpCy5.5, CCR5 FITC, CXCR4 APC (Biolegend, San Diego, CA) and Foxp3 PE.

CD4 PercpCy5.5, HIV-1 p24 FITC (Beckman Coulter, Brea, CA), Bcl-2 PE (BD Biosciences, San Jose, CA) and Foxp3 e-fluor660 (eBioscience).

CD4 PercpCy5.5, CD127 Pac Blue, Foxp3 PE, HIV-1 p24 FITC and IFN-γ Alexa Fluor 647 (Biolegend).

Isotype antibodies used included: IgG2a APC, IgG1 Pac Blue, rat IgG2a PE, IgG2a FITC, rat IgG2a AP, IgG1 FITC, IgG1 PE, IgG2a e-fluor660, and IgG1 Alexa Fluor 647.
The protocol and buffer set from eBioscience were used for all experiments involving intracellular staining. All samples were fixed and acquired within 1h of completion of staining, and analysis was by Miltenyi MACSQuant Flow cytometer.
Data were analyzed by FlowJo software (Treestar Inc, Ashland, OR) at the completion of study.
Hirsch C.S., Baseke J., Kafuluma J.L., Nserko M., Mayanja-Kizza H, & Toossi Z. (2016). Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection. Journal of clinical & experimental immunology, 1(1), http://www.opastonline.com/wp-content/uploads/2016/11/expansion-and-productive-hiv-1-infection-of-foxp3-positive-cd4-t-cells-at-pleural-sites-of-hivtb-co-infection-jcei-16-007.pdf.
+ Open protocol