FITC Rat Anti-Mouse CD4

Protocol tips

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Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).	Anti-CD4 FITC (clone H129.19, BD Biosciences) used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added.

Upstream tips
Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).
Protocol tips
Anti-CD4 FITC (clone H129.19, BD Biosciences) used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added.

Publication protocol

"Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).

To stain myeloid-derived suppressor cells (MDSCs), anti-CD11b FITC (clone M1/70, BD Biosciences) and anti-Gr-1 APC (clone RB6-8C5, BD Biosciences) were added to cells suspended in PBS with 3% FCS and incubated for 30 min. Anti-CD4 FITC (clone H129.19, BD Biosciences) and anti-CD25 PE (clone PC61, BD Biosciences) antibodies were used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added. To analyze CD4+ IFN-γ+ T cells and cytotoxic T lymphocytes (CTLs) expressing granzyme B, anti-CD4 FITC (clone H129.19, BD Biosciences), anti-CD8a PerCP (clone 53–6.7, BD Biosciences), and anti-IFN-γ APC (clone XMG1.2, BD Biosciences) were added to the cells and incubated for 30 min. Cells were then fixed and permeabilized, and intracellular staining was performed using an anti-granzyme B PE antibody (clone NGZB, eBioscience). All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA)."

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