Facsort cytometer

The intracellular cytokines were detected by flow cytometry. For intracellular staining, cells were permeabilized using a BD Fixation/Permeabilization kit (BD Bioscience). The antibodies used were FITC-conjugated anti-human IL-6 (eBioscience), PE-conjugated anti-human TGF-β1 (BioLegend, San Diego, CA, USA), and PE-conjugated anti-human IL-10 (eBioscience). Analyses were performed on a FACSort cytometer using CellQuest software (BD Bioscience). Immunotyping was detected according to our previous study [30 (link)].
To measure the secretions of human IL-6 and TGF-β1 on TNFα- treating MSCs, MSCs were treated with or without 10 ng/ml TNFα for 3 days. The concentration of these cytokines was measured in the supernatants using Platinum ELISA kits (eBioscience) and murine IL-10 and IL-17 ELISA kits (R&D Systems, Minneapolis, MN, USA). All of the samples from cocultured supernatants or serum were quantified according to the manufacturer’s instructions.

Lymph nodes were isolated from healthy male DBA/1J mice. The mice were euthanized by inhalation of CO₂, and the lymph nodes and spleen were harvested and crushed in phosphate-buffered saline (PBS). To isolate the cells, the suspension in 4 ml PBS was slowly placed on 8 ml Ficoll–Hypaque, the mixture was centrifuged at 500 × g for 30 minutes, and the cells were collected and suspended in RPMI 1640.
To determine the effects of MSCs on T cells, 10⁵ MSCs were treated with or without 10 ng/ml TNFα for 24 hours, and then stimulated with 1 × 10⁶ splenocytes with phytohemagglutinin-L (Sigma Aldrich) and 1 μg/ml anti-CD146 antibody in RPMI 1640 containing 10 % FBS. After 2 days, the suspended cells were harvested and Th17 and Treg cells were identified by flow cytometry. The supernatants from MSC–T cell cocultures were harvested and detected the cytokine levels for an ELISA assay. The antibodies used were fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD4 (eBioscience, San Diego, CA, USA), phycoerythrin (PE)-conjugated rat anti-mouse IL-17A (eBioscience), and PE-conjugated rat anti-mouse Foxp3 (eBioscience). Analyses were performed on a FACSort cytometer using CellQuest software (BD Bioscience).

Samples were analyzed using flow cytometry in a FACSort cytometer (BD, Franklin Lakes, NJ, USA) and Cell Quest (BD) and by FlowJo software version 10.2 (FlowJo, Ashland, OR, USA) [26 (link)]. Analysis of the immunoinfiltrate in mice lungs was performed by Flow cytometry of single cell suspensions. For this, lungs were collected, washed in PBS, minced and digested using Collagenase IV (Merck) at 37 °C for 30 min. After incubation, the remaining tissues were filtered through a 70 μm filter (BD) and washed in complete EMEM medium. After isolation by Ficoll density gradient, leukocyte were stained for flow cytometry analysis. Briefly, 0.3 × 10⁶ cells were incubated with anti-CD3-FITC, anti-NKp46-APC and anti-CD45-PerCP-Cy5.5 antibodies for 15 min in the dark, fixed (1x BD lysis solution), washed (PBS) and then were acquired in a LSR Fortessa X-20 Cytometer (BD). To analyse data, lymphocyte region was selected on the forward scatter versus side scatter plot and then gated on the CD45+ cells to exclude cell debris and non-hematopoietic cells. NK cells were identified as CD3−/NKp46+ cells.