Foxp3 pe clone nrrf 30

B and T cells were analyzed by flow cytometry (FACS) for expression of activation and differentiation markers. Briefly, single-cell suspensions were prepared from draining lymph nodes and spleen. Cells were resuspended in PBS with 10% FCS, counted (Nucleocounter) and seeded in 96-well plates. T cells were Fc blocked (clone 2.4G2 BD) before application of surface antibodies CD4 (V450 clone RM4-5, BD), CD4 (PB clone RM4-5, BD), CD25 (APC clone 3C7, BD) or CD25 (FITC, 3C7 BD) for 20 min in room temperature (RT). B cells were stained without prior Fc blocking using v450-labeled B220 (BD Biosciences) and APC-labeled CD93 for 20 min in RT. Intracellular staining was performed using FoxP3 / Transcription Factor Staining Buffer set (eBioscience) and FoxP3 (PE clone NRRF-30 eBioscience), FoxP3 (FITC, clone FJK-16s, eBioscience) or Helios (APC Alexa Fluor 647 clone 22F6 eBioscience) for 30 min in +8C°. Isotype controls were used as negative controls. Analysis was performed by FlowJo Software (Tree Star Inc., Ashland, OR, USA) and gates for surface staining were set according to flourochrome minus one [39 (link)].

SA1/N and B16-SIY tumor analyses were performed 2 days following the second dose of antibody treatment. Tumors were weighed on a microscale on plastic weigh boats and dissociated with a 0.25mg/mL Liberase TL (Sigma-Aldrich), 0.05mg/mL DNaseI (Roche) from bovine pancreas, and RPMI Medium mixture for 20 min at 37To determine ICOS receptor quantity on primary cells, tumors were processed as described above. Peripheral blood was collected by cardiac puncture with 25-gauge needles and tuberculin syringes and dispersed into EDTA coated blood collection tubes. RBCs were lysed in conical tubes with 1mL of ACK lysis buffer for 3min on ice. Blood was centrifuged at 400xg for 5mins and resuspended in 100uL FACS buffer. Tumors and peripheral blood were transferred to round bottom 96-well plates and Fc blocked as described above. Following Fc block, cells were stained with 1ug/mL DyLight-650 (Life Technologies) labelled ICOS antibody 37A10 or isotype control, in addition to the staining cocktail described above. Cells were stained in 100uL of cocktail for 30mins at 4ºC. Cells were then washed twice with FACS buffer and resuspended 100uL in Fix/Perm buffer (Foxp3/Transcription Factor Staining Kit, eBioscience) and kept at 4ºC for 15mins. Cells were washed twice in Perm buffer and resuspended in 100uL of intracellular staining cocktail containing fluorescent labelled anti-FoxP3 (FJK-16s, 1:100, eBioscience) diluted in Perm buffer. Following a 30min incubation at 4ºC, cells were washed twice in FACS buffer and resuspended in 100uL of FACS buffer supplemented with 0.1% paraformaldehyde. Receptor quantitation was performed using Quantum Simply Cellular beads (Bangs Laboratories) according to manufacturer’s protocol. To determine the number of cells per milligram of tumor, a fixed number of CountBrite beads (Life Technologies) were added to samples and analyzed by flow cytometry.
For TIL analysis of MC38 tumors, huCTLA-4 mice were randomized in groups when tumor reached an average size of 153mm³ and treated with a single dose of clinical grade ipilimumab (Yervoy®, Bristol-Meyers Squibb) or human IgG1 isotype control administered intravenously at 10 mg/kg, or a single dose of ICOS antibody or mouse IgG2a isotype control administered intraperitoneally at 0.25 mg.kg. Mice were sacrificed 72 hours later and tumors excised, dissociated, and processed for flow cytometry analysis. The following antibodies were used: CD45-BB515 (clone 30-F11, BD), CD3-BUV395 (clone 145-2C11, BD), CD4-APCeF780 (clone GK1.5, eBiosciences), CD8-PEeF610 (clone 53–6.7, eBiosciences), FoxP3-PE (clone NRRF-30, eBiosciences), CD25-PerCP/Cy5.5 (clone PC61, Biolegend), CD19-BV605 (clone 6D5, Biolegend), CD49b-APC (clone DX5, Biolegend), ICOS-BV421 (clone C398.4A, Biolegend). Viability was assessed by staining with Fixable Viability Dye eF506 (Biolegend).

The colon tissue selected from 0.5 cm below the cecum to 0.5 cm above the anus was cut longitudinally and then transversely into 0.5 cm pieces after removing adipose tissue, mesenteric connective tissue, and Peyer's patches. After washing with PBS, colon pieces were digested with collagenase solution containing 1 mg/mL collagenase VIII and 1 U/mL DNase I (Gibco, Life Technologies) for 55 min in a 37 °C shaker. The supernatant was filtered with 40 μm cell strainer, and centrifuged at 2000 rpm for 5 min. The cell suspension was centrifuged after washing with plain RPMI 1640. Lamina propria lymphocytes (LPLs) were separated by density gradient centrifugation (cells were resuspended with 40% Percoll solution, and overlaid with an 80% Percoll solution, at 2500 rpm, 25 min). The interface containing the LPLs were aspirated and washed in medium, and subjected to stain with anti-CD4 (clone GK1.5; eBioscience), anti-CD25 (clone PC61; eBioscience) at 4 °C in the dark for 30 min. Cells were fixed and permeabilized with 200 μL fixation-permeabilization buffer overnight at 4 °C in the dark. Subsequently, cells were incubated intracellularly with Foxp3-PE (clone NRRF-30; eBioscience) at 4 °C in the dark for 1 h, and were analyzed by flow cytometry (Cyto Flex S). The data were analyzed by FlowJo software (Tree Star).