Anti mouse cd3 pe cy7

Splenocytes (10⁷ cells/well) were cultivated in the presence of RBD (10 μg/mL) as a specific antigen or Con A (20 μg/mL) as a positive control. Unstimulated samples were used as a negative control. The stimulated cells were grown at 37 °C for 72 h and then incubated for an additional 5 h with Brefeldin A (BioLegend, San Diego, CA, USA). After incubation, the stimulated cells were harvested, treated with TruStain FcX (Anti-mouse CD16/32 antibody, BioLegend, San Diego, CA, USA) and subjected to surface staining with PE-Cy7 anti-mouse CD3, PE anti-mouse CD4 and FITC anti-mouse CD8 (BD Biosciences) antibodies. Subsequently, cells were processed with a fixation/permeabilization kit (BD Biosciences, San Diego, CA, USA), followed by staining with APC anti-mouse IFN-γ (BioLegend, San Diego, CA, USA). The percentages of positive cells were enumerated by flow cytometry. Simultaneously, aliquots of the culture supernatant were harvested at 12, 24, 48 and 72 h of treatment, and were then subjected to IFN-γ, IL-2 and IL-4 detection using ELISA (BioLegend, San Diego, CA, USA)

Immediately after the mouse livers were removed, cells were recovered by mechanical disruption, and immunostained for flow cytometric analysis. The mononuclear cells (MNCs) of liver sample were extracted with Percoll (GE Healthcare, Sweden). The cells were collected, washed twice in 0.2% bovine serum albumin (BSA) in PBS buffer (0.2% BSA-PBS) and resuspended in PBS buffer for counting cell numbers with Trypan blue stain (Gibco, United States). Anti-mouse CD16/32 (eBioscience, United States) was used as the purified blocking antibodies. Immuno-phenotyping was performed on the samples with the following anti-mice antibodies: anti-mouse NK1.1-APC, anti-mouse CD3-PE-Cy7, anti-mouse NKG2D-PE, anti-mouse NKp46-PE, PE Rat IgG1κ Isotype and PE Rat IgG_2aκ Isotype (BD PharMingen, United States). Data were acquired on BD LSR Foretessa flow cytometer (BD Biosciences, United States) and analyzed by using Flowjo 7.6 software.

For analysis of tumor infiltrating lymphocytes, resected tumor tissues were cut into small pieces and then digested in collagenase I (1 mg/ml) and 13.3 μl DNase I (50 U/ml) at 37 °C for 30 min. The mixture was filtered through a 70-μm strainer to prepare a single cell suspension. Cells were then washed twice with PBS and re-suspended in PBS, and 1 × 10⁶ cells were incubated with 3 μl antibody for 30 min at 4 °C in darkness. Wash the cells twice and perform the analysis on the FACSCalibur (BD Biosciences, USA). The anti-mouse CD3-PE-Cy7, CD8-FITC, CD45-APC-Cy7, F4/80-PE, CD25- PerCP-Cy5.5 and CD11b-BV650 antibodies were all purchased from BD Biosciences. Data are represented as the percentage of lymphocytes as indicated.

For analysis of tumor-infiltrating lymphocytes, resected tumor tissues were cut into small pieces and then digested in collagenase I (1 mg/ml) and 13.3 μl DNase I (50 U/ml) at 37 °C for 30 min. The mixture was filtered through a 70-um strainer to prepare a single-cell suspension. Cells were then washed twice with PBS and re-suspended in PBS, and 1 × 10⁶ cells were incubated with 3 μl antibody for 30 min at 4 °C in darkness. The cells were washed twice and performed the analysis on the FACSCalibur (BD Biosciences, USA). The anti-mouse CD3-PE-Cy7, CD8-FITC, CD45-APC-Cy7, F4/80-PE, CD25-PerCPCy5.5, CD4-Alexa Fluor 647, and CD11b-BV650 antibodies were all purchased from BD Biosciences. Data are represented as the percentage of lymphocytes from parents as indicated.

Flow cytometry analysis was performed using the following antibodies (BD Bioscience): Anti-mouse CD3-PE/Cy7, Anti-mouse CD11c-PE/Cy7, Anti-mouse CD8-PE/Cy5, Anti-mouse CD44-PE, Anti-mouse CD62L-FITC, Anti-mouse PD-L1-PE, Anti-mouse CD326 PE/Dazzle, Anti-mouse CD4-PE/Dazzle, Anti-mouse CD80-PE, Anti-mouse CD86-PE/Dazzle, and Anti-mouse CD45 FITC. All samples were suspended in FACS buffer and stained with 1 µL antibodies for 30 min at 4°C in darks, then washed twice and resuspended in FACS buffer before analysis. For γ-IFN detection, Human Cytometric Bead Array (CBA) kit and mouse CBA kit (BD Bioscience, USA) were used. The antibody dilution concentration was 1:100. Samples were analyzed with BD Accuri C6 (BD Bioscience, USA) and CytoFLEX (Beckman, USA).

Splenocytes and mesenteric lymph nodes (MLN) were collected at week 4 after challenge infection with T. gondii ME49. Collected cells were stained with fluorescence-labeled anti mouse CD3-PE-Cy7, CD4-FITC, CD8-PE, B220-FITC, and GL7-PE (BD, San Diego, CA, USA) antibodies in staining buffer (2% bovine serum albumin and 0.1% sodium azide in 0.1M PBS) after Fc receptor blocking as described previously [15 (link)]. Cell populations were acquired using Accuri C6 (BD, San Diego, CA, USA) and data was analyzed using the Accuri C6 software (1.0.264.21, BD Biosciences, San Diego, CA, USA).