Anti mouse cd4 pe

The splenocytes were prepared and cultured as previously described [27 (link), 28 (link)], and the splenocytes were plated in 6-well flat-bottom plates (5∗10⁶ cells in 2 mL of cRPMI per well) with 100 μL TB-PPD (1 μg/mL; XiangRui Biotech, Ltd., Beijing, China) in each well and incubated for 72 h (37°C, 5%CO₂). The cells were collected and washed three times with 0.1 M PBS (PH = 7.2), and then rabbit anti-Mouse CD4⁺-PE and anti-Mouse CD8⁺-FITC (eBioscience, USA) were added into EP tube for a 30 min incubation in an ice-bath keep out of the sun. Finally, the cells were washed twice again and the proportions of CD4⁺ and CD8⁺ T cells were determined by flow cytometry (FACSCalibur, BD).

Thirty-two mice were euthanized for evaluation of total and differential cellularity in bronchoalveolar lavage fluid (BALF), mediastinal lymph nodes, and thymus. First, animals were euthanized with a lethal dose (100 mg/kg) of thiopental sodium (Cristália). The trachea was immediately cannulated, then flushed three times with 0.4 mL of 1% PBS solution in order to obtain BALF cells. BALF was then centrifuged (239 g, 10 min) and the cell pellet was resuspended in PBS. Cells from the lung-draining mediastinal lymph nodes and thymus were obtained through mechanical maceration of the organs. Total leukocytes were quantified with Türk solution in a Neubauer chamber.
T cells recovered from BALF, lymph nodes, and thymus were distinguished after staining with anti-mouse CD4 PE (eBioscience, SanDiego, CA, USA) for T helper cells, anti-mouse CD8 PECy5 (eBioscience, SanDiego, CA, USA) for T cytotoxic cells, and anti-mouse anti-CD4-FITC, anti-mouse anti-CD25-APC, and anti-mouse anti-Foxp3-PE (Treg kit, eBioscience, SanDiego, CA, USA) for T regulatory (Treg) cells. These cells were evaluated in a FACSCalibur flow cytometer (Becton Dickinson Biosciences, San Jose, CA, USA) and analyzed in FlowJo 7.6.5 software (TreeStarInc., Ashland, OR, USA).

Handpicked islets were pooled in 24-well plates for 24 h in RPMI supplemented with 10% FCS, penicillin–streptomycin, and 5.10⁻⁵ M β-mercaptoethanol allowing extrusion of infiltrating lymphocytes (25 (link)). Infiltrating lymphocytes were recovered. Cells recovered were used for the analysis of T cells and antigen-presenting cells. T-cell analysis was performed using the Foxp3 intra-staining buffer set according to the recommendation of the manufacturer (eBioscience) with the following combination: anti-mouse CD45-efluo450, anti-mouse CD3ϵ-AlexaFluo700, anti-mouse B220-HorizonViolet500, anti-mouse CD8α-Percp-Cy5.5, anti-mouse CD4-PE, anti-mouse Foxp3-APC, and anti-mouse CD25-BrillantViolet711 mAbs (eBioscience). β Cells and dendritic cells were analyzed with anti-mouse CD45-AlexaFluo700, anti-mouse TCRβ-APC-Cy7, anti-mouse B220-HorizonViolet500, anti-mouse CD11b-efluor450, anti-mouse CD11c-APC, anti-mouse CD8α-BrillantViolet605, and anti-mouse CD4-PE mAbs (eBioscience). Acquisitions were performed with a BD LSR-Fortessa flow cytometer and analyzed using FlowJo 10.7.1 software.

Sections of the spleen, axillary lymph nodes, and tumors were prepared as single cell suspensions. The following flow cytometry antibodies were use: anti-mouse CD3 PE-Cyanine5 (15-0031-81; eBioscience), anti-mouse CD4 PE(12-0041-81; eBioscience), anti-mouse CD 8 FITC (12-0081-81; eBioscience), anti-mouse LY-6C APC (17-5932-80; eBioscience), anti-mouse LY-6G FITC (11-9886-80; eBioscience), anti-mouse F4/80 PE (565410; BD Pharmingen), anti-mouse CD11b APC-Cy7 (561039; BD Pharmingen), anti-mouse CD4 PE-Cyanine5 (15-0041-81; eBioscience), anti-mouse CD25 PE (12-0251-81; eBioscience), anti-mouse Foxp3 Alexa Fluor^® 488 (126405; Biolegend). Intracellular staining was performed after conducting fixation and permeabilization using Transcription Factor Buffer Set (424401; Biolegend). Cells were incubated on ice for 30 min before analyzed the samples using a FACS Canto II cytometer. The resulting data were processed using FlowJo (version 10.0.7) software.

For the “TagSort” strategy, 50,000 splenocytes were stained with anti-Mouse CD4-PE (eBioscience cat no. 12-0043-82) at room temperature for 30 min according to the manufacturer’s instructions. The stained cells were washed with ice-cold 1X PBS twice and pelleted down at 500×g, 4 °C, 5 min. Experiments were carried out following the procedures described in Supplementary Methods. DAPI and PE double-positive cells were sorted into a 384-well plate for library construction. For the “SortTag” strategy, CD4+ T cells were purified first from mouse splenocytes using the Naive CD4 T-Cell Isolation Kit, Mouse (Miltenyi, cat. no. 130-104-453) following the manufacturer’s instruction without the anti-CD44 depletion step. The purified CD4 T cells were processed according to the procedures described in Supplementary Methods.

A total of 2.5 μL of anti-mouse CD3-eFluor450, 0.625 μL of anti-mouse CD4-PE, 0.625 μL of anti-mouse CD25-APC (all from eBioscience, California, USA) were added to 100 μL of peripheral blood. Following incubation at room temperature in the dark for 15–30 min, 1 mL of erythrocyte lysis solution was added to the samples and incubated for 15–20 min. The cells were washed with 2 mL phosphate-buffered saline (PBS) and resuspended in 500 μL PBS. The cells were then analyzed using a Beckman Gallios flow cytometer (Beckman Coulter, Inc., California, USA).