Lsrii cytometer

Cells were resuspended in PBS-2% FBS, transferred to a 96 round-bottom well plate (10⁶ cells/well) and labeled with indicated combinations of antibodies (Table S1 in Supplementary Material) or matched isotype control antibodies (Table S2 in Supplementary Material) for 30 min at 4°C. Cells were washed twice in PBS-2% FBS and fixed with Cytofix/Cytoperm solution (BD Biosciences) for 15 min at room temperature.
Data were acquired, with an LSRII cytometer (Becton-Dickinson, Cochin CYBIO platform) and analyzed with Kaluza Software (Beckman-Coulter). As shown on Figure 1, penile lymphocytes were identified based on their forward and side scatter, after a cell-doublet exclusion step. Next, lymphocytes were identified as CD45⁺ cells, and immune cell populations were defined according to their phenotypic characteristics, namely, CD3⁻CD19⁺ for B cells, CD3⁺CD4⁺ for CD4⁺ T cells, CD3⁺CD8⁺ for CD8⁺ T cells, and CD3⁻CD56⁺ or CD3⁺CD56⁺ for NK and NKT cells, respectively. Finally, phenotypes, activation status, as wells as functions, were evaluated for each population as indicated.

After 2-week culture in α-MEM plus 10% FBS, mouse BMSCs were trypsinized, pelleted and then resuspended in PBS containing 2% FBS to a concentration of 2 × 10⁶ cells/ml. Freshly isolated bone marrow cells were resuspended to a concentration no more than 1 × 10⁷ cells/ml. Cells were stained with anti-mouse CD45 PerCP-Cy5.5 (eBioscience, 45-0451-80), anti-mouse/rat CD29 PE-Cy7 (eBioscience, 25-0291-80), anti-mouse CD105 (Endoglin) PE (eBioscience, 12-1051-81), and anti-mouse Ly-6A/E (Sca-1) APC (eBioscience, 17-5981-81), and analyzed with an LSRII cytometer (Becton Dickinson, San Jose, CA, USA). For BrdU Incorporation, the mice were intraperitoneally labeled twice (a 16-h interval) with 1 mg/mouse/injection BrdU and euthanized 2 h after the second injection. Freshly harvested bone marrow was treated with 1× RBC Lysis Buffer (eBiosciences, San Diego, CA, USA). The remaining cells were stained with anti-mouse CD45 PerCP-Cy5.5, anti-mouse/rat CD29 PE-Cy7, anti-mouse CD105 (Endoglin) PE, and anti-mouse Ly-6A/E (Sca-1) Alexa Fluor 700 (eBioscience, 56-5981-82) before permeabilization, DNase treatment and staining with anti-BrdU APC according to the instruction of APC BrdU Flow Kits (BD Pharmagen, Franklin Lakes, NJ, USA). The percentage of BrdU-positive fractions was assessed using FlowJo analysis software (Tree Star, Ashland, OR, USA).

Prior sacrifice, mice were anesthetized with isoflurane (1%), and blood was collected via retro-orbital puncture. One-hundred microliter of blood were subjected to automated blood parameter analysis using a VetScan hematology device (Abaxis, USA). Also, 100 µl of blood were incubated with TruStain FX (BioLegend, USA) to block Fc-receptors, and stained with ten different monoclonal antibodies (clone) directed against surface molecules of major leukocyte subpopulations: Ly6C FITC (HK1.4), NK-1.1 PE (PK136), CD11b PE-Dazzle (M1/70), CD8a PerCPCy5.5 (536.7), CD3 PECy7 (17A2), CD43 APC (S11), CD4 AlexaFluor700 (RM45), B220 APCCy7 (RA36B2), CD115 BV421 (AFS98), Ly6G BV510 (1A8); all BioLegend. Red blood cells were lysed using an ammonium chloride-based buffer (BioLegend), and pellets washed with FACS buffer. Samples were acquired on a three-laser, ten-color Gallios flow cytometer (Beckman-Coulter, USA). Data analysis was performed using Kaluza 1.5 software (Beckman-Coulter). For cytokine analyses, blood plasma was obtained by centrifugation, and stored at −80 °C until analysis using cytometric bead array (Becton-Dickinson, USA) which was acquired on a LSRII cytometer (Becton-Dickinson). Also, splenocytes were recovered from sacrificed mice and incubated in vitro overnight. Supernatants were taken off and analyzed using cytometric bead array.

Cell viability was determined by crystal violet staining (0.2% w/v in 2% ethanol) and Trypan blue or propidium iodide exclusion (GIBCO-Invitrogen, CA). For cell cycle analysis, MEFs were fixed in ice in 70% ethanol, washed with 1% BSA-PBS, incubated with 1 mg/mL RNase A for 10 min at 37°C, and then resuspended in PBS containing 0.1 mg/mL propidium iodide. Acquisition and analysis of DNA content was by flow cytometry using an LSR II cytometer (Becton Dickinson, NJ) and FlowJo software (Tree Star, OR). Apoptotic cell populations were determined by TUNEL assay (Roche, IN) according to the manufacturer’s recommendations with minor changes: cells were first trypsinized to make a single cell population, which was then fixed, permeabilized and labeled with dUTP-conjugated to FITC. The percentage of TUNEL positive apoptotic cells was determined by flow cytometry enumeration of fluorescent cells (FITC-A: 570–620 nm).

The APC BrdU Flow Kit (Becton Dickinson, Franklin Lakes, NJ, USA) was used for flow cytometric analysis of the cell cycle according to the manufacturer’s protocol. After the respective treatment (either cytokines, drugs, or controls), BrdU solution was added for another 3 h into the medium of the cells (10 µL of 1 mM BrdU per mL culture medium). Then, both the adherent as well as the detached cells in the supernatant were collected and counted. A total of 5 × 10⁵ cells/well were placed into a 96-deep-well plate, fixed, permeablized, and stained according to the manufacturer’s protocol. Flow cytometric measurements were performed on an LSRII cytometer in combination with the FACSDiva software V. 9.0, and the derived data were analyzed using the FlowJo software V. 10.8.0 (all from Becton Dickinson).