Facscallibur flow cytometer

The cytokines measured were TNF, IL-8, IL-10 and IFNγ. The Cytometric Bead Array (CBA) Human Flex Set system (BD Biosciences) provides a method of capturing a soluble analyte or set of analytes with beads of a known size and fluorescence, enabling the detection of analytes using flow cytometry. The detection reagent supplied in the kit is a mixture of phycoerythrin (PE)-conjugated antibodies, which emits a fluorescent signal proportionally to the amount of bound analyte. When the capture beads and detectors reagents are incubated with an unknown sample containing identified analytes, sandwich complexes are formed. Granzyme B, IFN-γ and TNF-α levels were quantified in culture media from macerated lesions from ten patients. Thirty microliters of all samples were prepared following CBA multiplex kit manual, and the cytokines were detected within a range of 10–2,500 pg/ml. The assays were performed according to the manufacturer’s instructions, and samples were acquired in an FACSCallibur flow cytometer (BD Biosciences). The data were analysed with FCAP Array Software version 1-01 (Soft Flow, Inc., St. Louis Park, MN, USA).

hDPSCs were characterized according cell morphology, stemness and proliferative mRNA marker expression, and surface marker analysis. Cell morphology was captured by phase-contrast microscope. RT-qPCR was used to analysed mRNA marker expression regarding stemness property (REX1, NANOG, and OCT4) and proliferative marker (Ki67). Cells were then characterized by flow cytometry for MSC surface markers. Briefly, the cells were stained with FITC-conjugated anti-human CD105⁺ antibody (Bio Legend, California, USA), FITC-conjugated anti-human CD73⁺ (Ecto-5′-nucleotidase) antibody (Bio Legend, California, USA), FITC-conjugated anti-human CD90⁺ (Thy1) antibody (Bio Legend, California, USA), FITC-conjugated anti-human CD44⁺ antibody (Bio Legend, California, USA), and FITC-conjugated anti-human CD45⁻ antibody (Bio Legend, California, USA). FITC-conjugated Mouse IgG1κ isotype control (FC) antibody (Bio Legend, California, USA) was used as an isotype control for this assay. The assay was performed by using FACScallibur flow cytometer with CellQuest software (BD Bioscience, New Jersey, USA).

THLE2 or CHO-K1 cells were harvested by trypsinization and centrifugation. After washing with PBS, cells were fixed in 4% paraformaldehyde at room temperature for 20 mins and washed with TBS three times. Without permeabilization, cells were blocked with 10% donkey serum in TBS for 10 mins, and incubated with rabbit anti-CKAP5 antibody (cat# HPA040375, Sigma-Aldrich) and rabbit IgG as a control in the blocking buffer at room temperature for 1 hour. Cells were washed with TBS three times and incubated with anti-rabbit PE conjugated secondary antibody (Thermo Fisher Scientific) for 30 mins. Cells were washed again with PBS buffer for three times and re-suspended in 1 ml PBS, and analyzed on a BD FACSCallibur flow cytometer (BD Biosciences) according to the manufacturer's protocol. Data are presented in the form of histograms.

Log-phase cultures (OD₆₀₀ ∼ 1.0) were pelleted and resuspended in 50 mM sodium citrate and sonicated to disperse cell clumps. Subsequently, the cells were treated with 250 µg/mL RNase A (Sigma-Aldrich, R6513) and 1 mg/mL Proteinase K (Sigma-Aldrich, P2308) overnight at 37°C. Finally, the cells were resuspended in 50 mM sodium citrate solution containing 1 µM SYTOX green (Thermo Fisher, 10768273). Samples were run on BD FACSCallibur flow cytometer equipped with a 15-mW 488-nm laser. Maximum count peaks for fluorescence intensities were calculated using the BD FACSDiva 8.0.1 software.