Anti cd3e fitc

Cells were incubated with an anti-CD16/CD32 monoclonal antibody (mAb, eBioscience, 14-0161-82, 93, 1:33) and then stained with the following dye or anti-mouse antibodies: viability dye APC-Cy7 (eBioscience, 65-0865-14), anti-lineage cocktail-FITC (BioLegend, 133302, 145-2C11; RB6-8C5; RA3-6B2; Ter-119; M1/70, 1:40), anti-TCRβ-FITC (BioLegend, 109206, H57-597, 1:200), anti-CD90.2-PE/Cy7 (BioLegend, 105326, 30-H12, 1:200), anti-CD127-APC (BioLegend, 135012, A7R34, 1:100), anti-ST2-BV421 (BioLegend, 145309, DIH9, 1:100), anti-CD3E-FITC (BioLegend, 100203, 17A2, 1:200), anti-TCRg-PE (BioLegend, 118107, GL3, 1:100). Gating was performed as follows: ILC2s were defined as live Lin⁻ (CD3e⁻Ly6G⁻Ly6c⁻CD11b⁻CD45R⁻TER119⁻TCRb⁻) CD127⁺Thy1⁺ST2⁺ cells. Stained cells were examined with a FACSCanto II flow cytometer (BD Biosciences), and the results were analyzed using FlowJo software version 10.4.2 (TreeStar, FlowJo).

Human PBMCs: For the apoptosis test, anti-AnnexinV-FITC (BD Biosciences, CA, USA) was used. To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA). For CCR7 expression, we used anti-CCR7-APC (Biolegend, CA, USA).
Murine lymphocytes: The phenotype of murine lymphocytes was analyzed by flow cytometry using anti-CD3e-FITC, anti-CD19-PB, anti-CCR7-APC, anti-CD44-FITC, anti-CD62L-APC, and anti-TCR-β-PB (Biolegend, CA, USA).
To eliminate the red blood cells, RBC lysis buffer (Beit Haemek, Israel) or BD FACS lysing solution (BD Biosciences, CA, USA) was added into murine blood and splenocyte samples. Flow cytometry was performed using the MACSQuant® Analyzer (Miltenyi Biotech, Germany), and the data were analyzed using FCS Express V3 software.

Briefly, the spleen and thymus were removed from anesthetized sixteen-week-old WT and KO mice to harvest lymphocytes for FACS analysis. These tissues were cut into small pieces with scissors and passed through a 70 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) to isolate a single-cell suspension. After removing the RBC from cell suspension using RBC lysis buffer (Sigma-Aldrich), the immune cells (1 × 10⁷ cells) were stained with PE/Cy7-anti-CD8 (100722; BioLegend, San Diego, CA, USA), FITC-anti-CD3e (152304; BioLegend), APC-anti-CD4 (100412; BioLegend), FITC-anti-CD43 (143203; BioLegend), APC-anti-CD45R/B220 (103211; BioLegend). Approximately 10,000 live cells were analyzed using a FACSCalibur™ Flow Cytometer (BD Biosciences). During these analyses, the stained cells were selected based on the forward and side scatter properties after gating for singlets. Each population was gated using the fluorescence intensity of the aforementioned antibodies against cell surface markers. The unstained cells were used to set appropriate negative gates by determining the background fluorescence levels. Single stained cells were used as a control to remove the spectral overlap between fluorophores.