Guava flow cytometer

mADSC were grown until confluent, trypsinized, and pelleted by centrifugation at 200 × g for 5 minutes. For fluorescence-activated cell sorting (FACS) analysis, ~1.0 × 10⁵ cells were resuspended in 100 μl FACS buffer containing 1 % fetal bovine serum in PBS. For FACS analysis of surface receptors, each sample was incubated for 30 minutes at 4 °C with FITC-conjugated, Alexa488-conjugated, PerCP/Cy5.5-conjugated, PE-conjugated, or Alexa Fluor-647-conjugated antibodies against the surface markers CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR5, CXCR6, and CXCR7 (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. After incubation, the labeled cells were diluted with 2 ml of FACS buffer, pelleted and resuspended in 300 μl of FACS buffer. Generally, ~5 × 10⁴ cells were analyzed per sample using the Guava flow cytometer (BD Biosciences, San Jose, CA, USA). Results were analyzed using GuavaSoft 2.7 software (BD Biosciences).

Leukocytes were isolated by lysing red cells with Ammonium Chloride (NH₄Cl, 0.149 mol/L; Riedel-de Haën, Sigma-Aldrich, Germany), then washed in PBS and used for phenotypic staining. Leukocytes were washed twice with PBS and then stained with conjugate anti-human monoclonal antibodies (mAbs): CD3 PerCP-Cy5.5 (clone UCHT-1), Vδ2 FITC (clone B6), CD56 FITC (clone NCAM16.2), CCR7 PE-CY7 (clone 3DI2), CD95 APC (clone DX2), CD69 APC-Cy7 (clone FN50), CD158a PE (clone HP-3E4) (BD Biosciences), CD16 PeCy7 (clone 3G8) (Beckman Coulter), and NKp46 APC (clone 9E2) (Milteny Biotech). Leukocytes were incubated with the cocktail of antibodies for 20 min at room temperature, then washed once with PBS and fixed with 8% Paraformaldehyde (PFA) for 30 min at room temperature in the pass through chamber. After fixation, cells were handled out the pass through and washed once with PBS. After washing, samples were immediately acquired with the GUAVA flow cytometer (BD Biosciences) in a BSL2 laboratory. A total of 100,000–200,000 events was acquired for each sample and analyzed with FlowJo 10.0 software (BD Biosciences). The gating strategy is shown in S1 Fig.