Macsquant running buffer

Surface marker staining was performed on enriched CD4+ T cells from PBMCs at day zero, CD4+ T cells co-culture with sMSCs, nsMSCs (direct contact and Transwell system), sHUV-EC-C cells, nsHUV-EC-C cells (direct contact and Transwell system) and CD4+ T cells cultured alone at day ten. Cells were pelleted down and resuspended in 100 μl staining buffer (containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA). Antibodies CD4-VioGreen (0.034 μg/ml), CCR10-PE (0.034 μg/ml), CD183 (CXCR3)-PE-Vio770 (0.02 μg/ml), CD194 (CCR4)-APC (0.068 μg/ml), CD196 (CCR6)-PE-Vivo-615 (0.013 μg/ml) were added and incubated for 10 min at 4 °C in the dark. Cells were washed with 1 ml staining buffer, pelleted and resuspended in 100 μL MACSQuant running buffer (Miltenyi Biotec). 5 μL of Propidium iodide (Miltenyi Biotec) was added shortly before running the samples on FACSARIA III (BD Biosciences). Data were analyzed with FlowJo software (v10, FlowJo LLC). To compensate optimally for fluorescence spillover from fluorochrome-conjugated antibodies, the MACS Comp Bead Kit, anti-human (130-104-187, Miltenyi Biotec) and anti-mouse (130-097-900, Miltenyi Biotec) Ig_κ(Miltenyi Biotec) were used.

Ca²⁺-fluorescence was measured as recently reported [31 (link)]. Briefly, 0.5 Mio PMNs/mL were centrifuged at 300× g for 10 min, following a resuspension of the cell pellet in 1 mL calcium buffer, containing final concentrations of 1 mM probenecid, 0.04% Pluronic^® F-127, and 4 µM of Ca²⁺-sensitive fluophor Fluo-4 AM. After incubation for 45 min at 37 °C and 5% CO₂, the cell suspension was washed twice with calcium buffer and centrifuged at 300× g for 10 min with a final resuspension of the cell pellet in 1 mL calcium buffer without Fluo-4. PMNs were stimulated with 50 µM MSG or 20 nM LY379268, with or without 100 nM mGluR2 antagonist 1 diluted in the same calcium buffer and incubated for 2 h at 37 °C and 5% CO₂. Following a centrifugation at 300× g for 10 min, the cell pellet was resuspended in 500 µL MACSQuant^® Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). Using a laser-guided flow cytometry, i.e., MACSQuant^® Analyzer 16 (Miltenyi Biotec, Bergisch Gladbach, Germany), the fluorescence was measured until 10,000 events were detected. For excitation and emission, wavelengths were 494 nm and 506, respectively. Data were analyzed using SigmaPlot 14.0 (Systat Software GmbH distributed from Inpixon GmbH, Düsseldorf, Germany). Solvent control (0.1% DMSO) was subtracted from each measurement.

AMt and NF cells, at second, fifth, and eighth passage, were harvested, and a total of 5 × 10⁵ cells from single-cell suspensions were dispensed per each tube. Samples were incubated for 30 min at 4 °C with labeled monoclonal antibodies (mAbs) specific for EpCAM-APC and CD90-FITC (Miltenyi Biotech, Bergisch Gladbach, Germany). Then, samples were washed twice and resuspended in MACSQuant™ Running Buffer (Miltenyi Biotech, Bergisch Gladbach, Germany). The flow cytometer was set using cells stained with isotype-identical antibody controls. Cells were gated on a forward (FSC) versus side scatter (SSC) plot in order to eliminate debris. Acquisition was performed collecting 10,000 events that were analyzed by MACSQuant^® Flow Cytometer using the MACSQuantify^® Software (Miltenyi Biotech, Bergisch Gladbach, Germany).

CD4⁺ and CD8⁺ differentiation of T cells from stimulated splenocytes as similarly described above in lymphocyte proliferation were incubated with CD3e-PE, CD8a-FITC, and CD4-PerCPVio700 antibodies (Miltenyi-Biotec, Bergisch Gladbach, Germany) at 4 °C for 30 min in dark. Additionally, to compare the induction of MHCI and MHCII molecules, FITC labeled MHC class 1 (H-2Kb) and MHC class II (I-A/I-E) FACS analysis markers (Bio-Rad Laboratories, Inc., Hercules, CA, USA) were used according to the manufacturer’s protocol. Cells were washed and resuspended with MACSQuant running buffer (Miltenyi-Biotec, Bergisch Gladbach, Germany) before flow cytometric analysis. The percentage of CD3⁺CD4⁺, CD3⁺CD8⁺ T cells, MHCI, and MHCII was analyzed using a benchtop flow cytometer, MACSQuant^® analyzer (Miltenyi-Biotec, Bergisch Gladbach, Germany).
Moreover, stimulated splenocytes in 24-well plates were harvested and total RNA was isolated. cDNA was then synthesized using reverse transcription master premix (Elpis Biotech, Daejeon, Republic of Korea) with oligo d(T)15 primer according to the manufacturer’s protocol. Levels of IFN-γ, TNF-α, and IL-4 mRNA were analyzed by qPCR using the primers listed in Table 1. The relative cytokine expression levels were determined by the 2^−∆∆CT method using β-actin as the housekeeping gene.

Freshly isolated MPCs and P2-MSCs were washed in MACSQuant™ Running Buffer (Miltenyi Biotech, Bergisch Gladbach, Germany) and stained with anti-CD11c VioBlue®, anti-CD18 APC, anti-CD31 PE, anti-CD34 VioBlue®, anti-CD45 APC-Vio770, anti-CD73 PE, anti-CD90 FITC, anti-CD133 APC, anti-CD146 FITC, HLA-DR VioBlue® (Miltenyi Biotech), anti-STRO-1 FITC, and CD144 PE (Biolegend, San Diego, CA, USA). Samples were acquired by MACSQuant® Flow Cytometer and analyzed by MACSQuantify® Software (Miltenyi Biotech).

For cell cycle analysis, cells were trypsinized and 0.5 × 10⁶ cells per sample fixed and permeabilized in 1 mL ice-cold ethanol (70%) at least 1 h at 4 °C. After fixation, the cells were centrifuged for 5 min at 150× g, washed with PBS, and incubated with 25 µL RNase A (1 mg/mL; Life Technologies, Carlsbad, CA, USA) in 500 µL PBS for 30 min at 37 °C. Following further centrifugation and washing steps, cells were carefully resuspended in 500 µL FACS buffer (MacsQuant Running Buffer, Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with 25 µL propidium iodide (1 mg/mL in PBS, Sigma–Aldrich, Steinheim, Germany) with gentle vortexing. Samples of 5000 cells were analyzed using flow cytometry (MacsQuant, Miltenyi Biotec, Bergisch Gladbach, Germany).

MNCs from the three above sources (150,000 cells per sample) were incubated with REAfinity^® anti-human CD64 (clone REA978) fluorescein isothiocyanate–conjugated, CD31 (clone REA730) PE/Cy7-conjugated, CD14 (clone REA599) VioGreen^® -conjugated, and CD45 (clone REA747) VioBlue^® -conjugated antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30’ at 4°C in the dark, and washed twice in MACS Quant^® Running Buffer (Miltenyi Biotec). Data were acquired using MACS Quant^® flow cytometer and analyzed by MACS Quantify^® Analysis Software (Miltenyi Biotec).
Flow cytometry of freshly detached cells from primary cultures was performed as described above using antihuman CD90 (clone DG3) FITC-conjugated, CD73 (clone AD2) PE-conjugated, CD31 PE/Cy7-conjugated, CD14 VioGreen^® -conjugated, and CD45 VioBlue^® -conjugated antibodies (Miltenyi Biotec).
Frequencies of cell populations were calculated on total events, after exclusion of cell debris on FSC vs. SSC density plots and doublets on FSC-A vs. FSC-H. Non-parametric Wilcoxon test for unmatched pairs was performed applying GraphPad Prism^® software (GraphPad Software, San Diego, CA, United States).