Cell quest software

To quantify the percentage of hematopoietic stem cells (HSCs), BM cells (1 × 10⁵ cells) were stained with anti-mouse CD117 (c-Kit) FITC and anti-mouse Ly-6A/E (Sca-1) PE antibodies (eBioscience, San Diego, CA, USA). The percentages of CD3⁺CD4⁺ (helper T cell, Th) and CD3⁺CD8⁺ (cytotoxic T cell, CTL) lymphocytes were also measured. After 12 h or 24 h of treatment, BM cells were harvested and stained with anti-mouse CD3 PE-Cyanine7, CD4 PE-Cyanine5 and CD8a PE antibodies (eBioscience, San Diego, CA, USA). The percentages of staining cells were counted and analyzed by FACS Calibur cytometer and CellQuest software (Beckman Coulter, Brea, CA, USA). Each experiment was performed in triplicate with three replicates each.

Expression of cell surface CXCR4 on MSCs was detected with PE-conjugated monoclonal anti-mice CXCR4 antibody (Biolegend, China). Briefly, cells were resuspended in 1 × Binding Buffer at a concentration of 10⁶ cells/ml. Five microliters of PE-conjugated monoclonal anti-mice CXCR4 antibody were added and incubated with cells at room temperature for 15 min. Labeled cells were then analyzed by a Cytoflex S flow cytometer with the use of Cell Quest software (Beckman Coulter, USA).

BMCs were obtained by femoral cavity flushing and filtered through a 200-eye cell sieve mesh to obtain a single-cell suspension. To quantify the percentage of HSCs, CD117- and sac-1-positive cells were washed and stained with anti-mouse CD117 (c-Kit) FITC and anti-mouse Ly-6A/E (Sca-1) PE antibodies (eBioscience). For the detection of apoptosis in BMCs, an Annexin V-FITC Apoptosis Detection Kit (eBioscience) was used. BMCs were stained with FITC-conjugated annexin V and propidium iodide (PI). Flow cytometry was performed using a fluorescence-activated cell sorter Calibur cytometer and analyzed with CellQuest software (Beckman Coulter, Brea, CA, USA).

When transfected siRNA with or treated with conditioned media for 48 h, HRMCs were collected, washed with PBS, and then fixed with 75% ethyl alcohol at 4 °C overnight. The following day, the ethylalcohol was removed, and the cells were washed with PBS two times, then fixed with 500 µl of propidium iodide (PI) staining solution and incubated for 30 min in the dark at 4 °C. The analysis of the cell cycle was performed with a FACSCalibur flow cytometer with CellQuest software (Beckman).

Detection of Strol-1⁺, the cell surface marker, was performed by trained technicians blinded to patient identity using Becton Dickinson FACS Calibur Flow Cytometry System (Becton Dickinson, Beckman Coulter, Brea, CA, USA) equipped with Cell Quest software (Beckman Coulter). BMSC cell suspension was incubated with primary antibody for 1 h at 4 °C. Unbound antibodies were removed by washing with PBS. The secondary monoclonal antibodies conjugated with allophycocyanin (APC) were used to detect Stro-1⁺ (BD Pharmingen, San Diego, CA, USA) (1:100 dilution). After incubation, cells were washed and resuspended in 500 L of wash buffer and measured by FACS. The signals corresponding to debris and cell aggregates were first gated out by using the forward light scatter (FSC) and side light scatters (SSC) display. Furthermore, absolute counts of Stro-1⁺ positive cells in BMSC were determined using BD TruCOUNT Tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. During analysis, the absolute number of Stro-1+ positive cells in cultured BMSC was manually calculated using the following equation: (events of Stro-1⁺ positive cells/events of beads) * (number of beads per test/test volume).

flow cytometry was used to measure the proportion of CD4+ and CD8+ lymphocyte subtypes in peripheral blood in each group, and we calculated the ratio of CD4+/CD8+. Peripheral blood (100 μl) was placed in EDTA in a labelled tube, and 20 μl of fluorescent anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies was added at 37°C for 20 min in the dark. After haemolysis, samples were centrifuged for 10 min at 1500 rpm at room temperature, washed twice in PBS, and subjected to flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) to analyse T lymphocyte subsets (CD4+ and CD8+). The percentage of CD4+ and CD8+ T cells was determined using Cell-Quest software (Beckman Coulter).