U bottom plate

The EAI assay was performed as described in detail elsewhere [9 (link)]. In brief, erythrocytes obtained from Balb/cJRj mice were purified using a Ficoll gradient (Ficoll-Paque, VWR, Cat#17-1440-02) and were stored for up to 2 weeks in DMEM (Gibco, Cat#61965) containing 10% FCS FCS (Amimed, Cat#2-01F30I) at 4°C prior to use.
Serial dilutions of sera were performed in 96-well U-bottom plates (Greiner, Cat#650161) in DMEM containing 10% FCS. 5x10⁵ cfu B. taylorii expressing GFP were added per well and the plates were incubated at 35°C, 5% CO₂ for 1h prior to the addition of 10⁶ red blood cells (multiplicity of infection, MOI = 0.5) in 100 μl DMEM containing 10% FCS. The next day, the supernatant was removed, the red blood cells were fixed using 1% PFA (EMS, Cat#EMS-15710) and 0.2% gluturaldehyde (EMS, Cat#16020) in PBS (BioConcept, Cat#3-05F29-I) for 10 min at 4°C in the dark. After quenching with 2% FCS in PBS, the cells were analysed for GFP signal by Flow Cytometry (BD Canto II using HTS autosampler).

Mouse splenocytes were isolated as described above. 5 × 10⁵ splenocytes were plated in 96-well U-bottom plates (Greiner) in R10 medium and incubated in the presence or absence of F(ab’)₂ fragments with 0.001, 0.01, and 0.1 μg/ml dried, inactivated M. tuberculosis (Des. H37 Ra, Difco Laboratories) suspended in R10 medium for 12 h under SCCC.

Proliferation assays were performed in 96‐well U‐bottom plates (Greiner Bio‐One). BMDCs (pulsed with or without lysates) were co‐cultured with 10⁵ CD3⁺ cells at different ratios (1:1, 10:1, and 100:1 CD3:BMDC) in RPMI 1640 medium as described above. The CD3⁺ cells were previously labeled with CellTrace Violet per the manufacturer's directions (Thermo Fisher Scientific). After 6 days of co‐culture, flow cytometry was used to measure CD3⁺ T‐cell proliferation. Supernatants were collected from cells co‐cultured for 24 h, and assayed by ELISA for interferon‐γ (Legend Max, BioLegend™).
Cytotoxicity assays were performed using 10⁵ rat splenic CD3⁺ cells co‐cultured in 96‐well U‐bottom plates with 10⁴ BMDCs in RPMI 1640, supplemented as described above. On day 7, the CD3⁺ cells were collected and incubated for 18 h in flat‐bottom 96‐well plates, along with target cells, at effector‐to‐target ratios ranging from 20:1 to 0.625:1. The plates were assayed for lysis by LDH assay (Thermo Scientific Pierce). CD107a expression was measured essentially as described by Betts et al (2003). BMDCs were added to CD3⁺ cells, co‐cultured for 1 h in the presence of CD107a antibody, and incubated for another 5 h in the presence of monensin and brefeldin A (BioLegend). The cells were fixed and stained with anti‐CD4, anti‐CD8, and secondary to CD107a antibodies and analyzed by flow cytometry.

In our preliminary studies, two UC-MSC : PBMC cell ratios were tested, 1:2.5 and 1:5. CD4⁺/CD8⁺ ratio, CD4⁺Ki67⁺ and CD8⁺Ki67⁺ percentages were tested, whereby the results using the 1:5 UC-MSC : PBMC ratio were more outspoken (data not shown). Therefore, the following experiments were performed with the 1:5 ratio (UC-MSC: PBMC or UC-MSC: CD4⁺).
100.000 CD4⁺ T cells were seeded in 96 well U-bottom plates (Greiner Bio-One) with complete RPMI medium. CD4⁺ T cells were activated with αCD3/CD28 beads (Dynabeads™ Human T-Activator, Gibco®Thermo Fisher Scientific) in 1:5 (bead-to-cell) ratio. After 48 h of culture 20.000 UC-MSC were seeded in 96-well U-bottom plates with complete MSC growth medium. Both CD4⁺ T cells and UC-MSC were kept in culture for 24 h to allow for activation and attachment, respectively. The following day, growth medium of UC-MSC was removed, CD4⁺T cells were resuspended in complete RPMI medium and transferred onto seeded UC-MSC. Cells were cultured together for up to 5 days, and flow cytometry measurement was acquired every other day.

Fresh spleens were collected aseptically and homogenized using the top of a syringe to gently pass through a 70 μm nylon cell strainer. The collected cell suspension was washed and incubated with lysis buffer (Thermo Fisher, Netherlands), for 5 min to remove red blood cells. Splenocytes were washed and resuspended in RPMI 1640 medium (Lonza), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bodinco, Alkmaar, Netherlands), penicillin (100 U/mL)/streptomycin (100 μg/mL; Sigma-Aldrich) and β-mercaptoethanol (20 μM; Thermo Fisher Scientific). Total cell number was determined by using a Beckman Z1 coulter^® Particle Counter (Beckman, United States) and splenocytes (10⁷ cells/ml) were cultured in 96-well U-bottom plates (Greiner Bio-One B. V., Netherlands), either with medium or with 0.2 μg/mL anti-CD3 (Thermo Fisher, Netherlands). After 4 days stimulation, the supernatants were harvested and stored at -20°C until further analysis. The concentrations of MCP-1, IL-17A, IL-13, IL-22, and IFN-γ were measured using Bead-based immunoassays (Procartaplex, Thermo Fisher, Netherlands) according to the manufacturer’s instructions.