PerCP Rat Anti-Mouse CD8a

Protocol tips

Upstream tips	Protocol tips	Downstream tips
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).	The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added.	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)."

Upstream tips
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).
Protocol tips
The cells were then fixed and permeabilized for intracellular staining, and an anti-Foxp3 APC (clone FJK16s, eBioscience) antibody was added.
Downstream tips
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)."

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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