Cd69 bv605

Single cell suspensions from mouse experiments were stained with Live/dead Aqua (Molecular Probes, Inc.), CD4-PerCP, CD8-Pacific Blue, CD69-BV605, CD62E-PE, CD62E-BV421, CD62P-Alexa647, CD162-AlexaFlour647, HECA454-PE (BD Bioscience), CD25-APC, CD31-BV605, CD105-Pacblue, I/A-I/E-BV421, CD45-APCCy7, EpCAM-APC-Cy7, Podoplanin-PE, CD31-PECy7, EpCAM-FITC, CD64-BV711 (Biolegend), CD11c-PECy5.5 (Invitrogen) CXCR3-PECy7, CD3-Alexa700 and Ly6C-efluor450 (eBioscience). For subsequent detection of CXCL10 mRNA Primeflow^® RNA Assay (eBioscience) was used accordingly to manufacture protocol. Samples were acquired on an LSR-II flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Single cell suspension isolated from human tumors and unaffected colon tissue were stained with Live/dead Aqua (Molecular Probes, Inc.), CD31-Alexa700, (Biolegend), CD4-PerCP, CD8-BV711, CD105-APC, CD14-Alexa700, CD19-APCH7 and CD19-PE-CF594 (BD bioscience) followed by permeabilization with Fix & Perm kit (ADG Bio research GMBH) and staining with CXCL9-FITC (R&D) and CXCL10-PE (Biolegend), flow cytometry analyses were performed as above.

An amount of 2 μg of anti-mouse CD45-BV786 antibody (BD Biosciences, Franklin Lakes, NJ, USA) was diluted in sterile PBS and administered intravenously in the tail vein 10 min before anesthesia. CD 45+ is a circulating lymphocyte, and CD45− is a tissue-resident cell. In total, 40 U/mL DNase I and 1 mg/mL Collagenase IV enzyme were measured to a volume of 2 mL and incubated with lung tissue at 37 °C for 30 min. The tissue was then homogenized using a dissociator, filtered through a 70 μM cell strainer, and subjected to RBC lysis to obtain single lung cells. After preparing the lung cells to a concentration of 5 × 10⁶ cell/mL, they were washed with PBS and live-cell staining was performed using fixable viability stain 700 (FVS700) (BD Biosciences, Franklin Lakes, NJ, USA). After treating lung cells with Fc Block^TM reagent, the surface markers CD44-BV421, CD69-BV605, CD103-PE, CD62L-APC, CD4-FITC, CD3-PE-Cy7, and CD8-BB700-per cycle 5.5 ((BD Biosciences, Franklin Lakes, NJ, USA)) were used for staining. The flow cytometry gating strategy was performed according to Supplementary Materials Figure S1, based on a previous study [45 (link)]. FACS Aria Fusion from BD Biosciences was used, and all results were derived using FlowJo software v10.