Facsariatm

Increasingly evidence indicated that the immunomodulatory function of MSCs is the basis for the treatment of systemic lupus erythematous (SLE), osteoarthritis, and other diseases. It is recommended as a potency as well a release standard for advanced period clinical trials by the ISCT [24 (link)]. The detailed experimental reagents and methods could be referred to our previous research [22 (link)]. Briefly, the immunomodulatory effects of HUCMSCs on Th1 (CD3⁺ CD8^- IFN-γ⁺), Th17 (CD3⁺ CD8^– IL17A⁺), and Tregs (CD4⁺ CD25⁺ Foxp3⁺) were detected by co-culturing HUCMSCs with human peripheral blood mononuclear cells (PBMCs) in our evaluation system. Flow cytometry (BD facsariatm, USA) was used to analyze the cells, and the data was analyzed by FACS software. The detailed experimental procedures described in the supplemental file. The inhibition of Th1 or Th17 proliferation was calculated as [[1 − (The percentage of Th1 (or Th17) in MSC group)/(The percentage of Th1 (or Th17) in PBMC group] × 100%]. The calculation method of Tregs promoting proliferation was as follows: [(The percentage of Tregs in MSC group − The percentage of Tregs in IL2 stimulation group)/The percentage of Tregs in IL2 stimulation group].

Single-cell suspensions of tumor tissue (as described above) and splenic monocyte suspensions (as described above) were blocked with PBS containing 5% BSA for 20 min in room temperature. Subsequently, tissue single-cells were stained with anti-mouse CD45-APC mAb (1:200 dilution; eBioscience™, RRID: AB_469392), anti-mouse CD8-FITC mAb (1:100 dilution; eBioscience™, RRID: AB_464915) and anti-mouse CD31-PE mAb (1:100 dilution; eBioscience™, RRID: AB_465632); Splenic monocytes were stained with anti-mouse CD45-APC mAb (1:200 dilution; eBioscience™, RRID: AB_469392), anti-mouse CD3-FITC mAb (1:100 dilution; eBioscience™, RRID: AB_2572431). After 30 min of staining, the cells were washed twice with PBS and re-suspended in 0.5% BSA (in PBS). Sorting was performed on a Fusion cell sorter (BD FACSAria^TM, USA). In the FSC/SSC-gated monocytes, the CD45⁺CD8⁺ single-cells of tumor tissue were recognized as tumor-infiltrating CD8⁺ T cells, the CD45^-CD31⁺ single-cells of tumor tissue were recognized as vascular endothelial cells, the CD45⁺CD3⁺ splenic monocytes were recognized as splenic T cells.

The cells were seeded in 60 mm plates and transfected with siRNAs 24 h later. The cells were irradiated with 10 Gy or treated with the indicated doses of CPT and incubated during 12 h before harvesting. In the experiments performed with protein inhibitors, the medium was exchanged 2 h before irradiation with fresh DMEM containing 10 μM ATMi (KU55933), 5 μM ATRi (ETP46464), 10 μM DNA–PKi (NU7441), or DMSO as control. After that, the medium was replaced, DSBs were induced with the indicated DNA damage agent, and cells were incubated for 10 h. Then, cells were harvested with trypsin, spun down at 500 g for 5 min and washed with PBS. The cells were fixed with 4% paraformaldehyde for 15 min at 4 °C in the dark and later rinsed and resuspended in 150 μl of PBS. Red and green fluorescence was measured on BD FACSAriaTM using FACSDiva v5.0.3 software as indicated in the above section.

Vectors containing target fluorescent genes ZsGreen, ZsYellow, DsRed, AsRed, mCherry, and mRFP (Takara Bio), were either transfected into B16-F10 cells using the calcium chloride precipitation method (all FPs except ZsGreen), or retrovirally transduced into B16F10 cells (ZsGreen). Transformed cells were then harvested, washed, and repeatedly sorted through fluorescent activated cell sorting (FACS) on a BD FACSAria^TM to select for stable fluorescent positive populations. Sorted cells were then collected, frozen and/or propagated for use in future experiments. Cells were cultured at 37°C with 5% CO₂ in DMEM (GIBCO) plus 10% heat-inactivated FCS with penicillin, streptomycin, and L-glutamate on tissue-culture treated plastic plates and split every other day.

Spleens and livers were harvested into ice cold FACS buffer (PBS supplemented with 2% FCS v/v) and then emulsified through a 70 µM filter (BD Biosciences), centrifuged and washed in FACS buffer. Liver mononuclear cells were isolated using isotonic percoll gradient. Tissue samples were treated with Red Blood Cell Lysis Buffer (BD Pharmlyse, BD Biosciences). Samples were incubated in CD16/CD32 blocking antibody (clone 93; Biolegend) before staining with appropriate fluorochrome-conjugated antibodies at the listed dilution (Supplementary Table 2). Post-acquisition analyses were performed using FlowJo software V10.0 (Treestar, CA). Cell analysis and sorting were performed using the BD FACS LSR Fortessa^TM or BD FACSAria^TM using FACS Diva Software (version 8.0.1).

With informed consent, the hUC-MSCs were isolated from fresh umbilical cord of a full-term delivery donor. The entire procedure was approved by the Medical Ethics Committee of The Affiliated Drum Tower Hospital of Nanjing University Medical School. The umbilical cord was washed with sterile PBS and was cut into 3–5 mm long pieces, suspended in Dulbecco's modified Eagle's medium–low glucose (DMEM-LG, Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). Then the isolated cells were cultured in an atmosphere of 5% CO₂ at 37 °C and the medium was replaced every 3–4 days until well-developed colonies of fibroblast-like cells appeared. The cells were then digested with 0.25% trypsin and seeded in new culture bottles for further expansion. The cells of fourth generation were harvested as purified hUC-MSCs and taken for further studies. The adhesiveness properties were identified and the morphological characteristics of hUC-MSCs were detected by an inverted microscope (Olympus, Japan). Flow cytometry (BD facsaria^tm, USA) was employed for hUC-MSCs characterization according to published protocols [23 (link)], data analysis was performed using FACS software.