Fitc conjugated antibody

Phenotypic analyses of cultured hMBSCs were performed using standard flow cytometry methods. Passage 3 hMBSCs were collected in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lakes, NJ) at a concentration of 1 × 10⁶ cells/ml in stain FACS buffer (PBS containing 2% FBS), and then stained with FITC-conjugated antibodies against human CD29, CD90, CD45, HLA-DR, CD80, and CD40, phycoerythrin- (PE-) conjugated antibodies against human CD73, CD105, CD34, HLA-ABC, and CD86, and their isotype controls (all from BD Biosciences) at 4°C for 30 min in the dark. After washing twice, the cells were resuspended in 200 μl of PBS and acquired by a FACSCalibur instrument (BD Biosciences). Dates were analyzed using the FLOWJO™ software (TreeStar, Inc., Ashland, OR, USA).

For flow cytometry, cells were incubated in BrdU labeling reagent (Invitrogen, Frederick, MD) for 1hr. Harvested cells were stained for 1hr with purified FITC-conjugated antibodies (BD Biosciences, San Jose, CA). Data were acquired on a FACScan fluorescence activated cell sorter (BD Biosciences) and analyzed using FlowJo software (Treestar Inc., Ashland, OR).

Cells were treated with a trypsin-EDTA solution. Cells were centrifuged at 1500 r.p.m. for 10 min and supernatants were discarded. Twenty microliter of PE-conjugated or FITC-conjugated antibodies(BD Pharmingen, San Diego, CA, USA) was then added to individual tubes and tubes were left in a 4 °C refrigerator in the dark for 30 min. Cells were washed twice with PBS. Cell pellets were re-suspended in 300 µl of PBS and analyzed with MACSQuant (Miltenyi Biotech Inc, Bergisch Gladbach, Germany). Isotype controls were included in all flow cytometry analyses. After cells were incubated for 30 min with the respective antibodies, propidium iodide staining was used to observe whether the number of dead cells increased. Flowjo v7.6.2 software (Treestar Inc, San Carlos, CA) was used for data analysis.

Phenotypic analyses of cultured hAMSCs were performed using standard flow cytometry methods. Passage 3 hAMSCs were collected in fluorescence-activated cell sorting (FACS) tubes (BD Biosciences, Franklin Lakes, NJ) at a concentration of 1 × 10⁶ cells/ml in stain FACS buffer (PBS containing 2% FBS) and then stained with FITC-conjugated antibodies against human CD29, CD90, CD45, HLA-DR, CD80, and CD40; phycoerythrin (PE)-conjugated antibodies against human CD73, CD105, CD34, HLA-ABC, and CD86; and their isotype controls (all from BD Biosciences) at 4 °C for 30 min in the dark. After washing twice, the cells were resuspended in 200 μl of PBS and acquired by a FACSCalibur instrument (BD Biosciences). Data were analyzed using FLOWJO TM software (TreeStar, Inc., Ashland, OR, USA).

MSCs at passage 3 were digested and resuspended in 100 μL of PBS with a density of 1 × 10⁵ cells. The cells were labeled with FITC-conjugated antibodies (BD Biosciences, San Diego, USA), including CD13, CD34, CD45, CD73, CD90, and CD105. FITC-conjugated isotype-matching IgS was used to determine nonspecific staining. The cells were examined using a FACSCalibur cytometer (BD Biosciences), and the data were analyzed using Cytosoft version 5.2 (Guava Technologies, Hayward, CA, USA).

The cells from six groups were analyzed for positive expression of MSC-specific markers (CD44, CD90, and CD105) and negative expression of a hematopoietic marker (CD45) using flow cytometry (BD FACS Calibur, BD), as previously described [7 (link), 19 (link)]. The cells were harvested, filtered through 40 μm meshes, fixed with 4% paraformaldehyde at 4°C overnight, and then blocked with 1% bovine serum albumin. The 1 : 200-diluted fluorescein isothiocyanate (FITC)-conjugated antibodies (BD) related to anti-CD44, anti-CD45, anti-CD90, anti-CD105, and an isotype control were labeled directly at room temperature (RT) for 1 h. Thereafter, 1 × 10⁴ FITC-labeled cells were counted with a flow cytometry.

4x10⁵ peptide-specific T-cell lines were stimulated with SCoV-DP15 in a final concentration of 20µg/ml for 12-14 hours. The stimulation was then carried out for an additional 4 h in the presence of 2mM Monensin (Biolegend, San Diego, CA, USA), 10ug/ml Brefeldin A (Biolegend) as well as FITC Conjugated antibodies to the granular membrane proteins CD107a (BD Biosciences, Heidelberg, Germany). Immediately following stimulation, cells were washed once and stained with LIVE/DEAD™ Fixable Aqua dye (Thermo Fisher Scientic, Waltham, MA, USA) prior to surface staining with the following anti-human monoclonal antibody reagents: anti-CD3 (AF700; BD), anti-CD4 (APC-F75; Biolegend), anti-CD8 (PerCP-Cy5.5; Biolegend) and anti-CD45RO (BV650; Biolegend). Next, cells were washed and then fixed with 2% paraformaldehyde then permeabilized with 0.5% saponin prior to staining with anti-γ-IFN (PE-Cy7; Biolegend). Finally, cells were washed and measured on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) prior to data analysis using FlowJo (Becton, Dickinson and Company. Ashland OR, USA). The gating strategy is described in Supplementary Figure S1.