Guava flow cytometer

C. albicans SC5314 cells were grown at 30°C until reaching exponential phase, diluted to ~1.2–3×10⁶ CFU/mL, and treated with SH1009 at the IC₅₀ concentration followed by incubation for an additional 3 h. After harvesting by centrifugation, supernatants were discarded and pellets were washed with PBS and then fixed with cold 70% ethanol at -20°C for 2 h. Fixed cells were washed in PBS and resuspended in 500 uL of PBS containing 20 μg/mL RNase and incubated at 37°C for 2 h. 200 μL of PBS containing 20 μg/mL propidium iodide (PI) were added to the treated cells. Using the Millipore Guava flow cytometer, 5000 events were counted, and the fluorescent intensity of PI measured. After acquiring the data using Guava PCA-96 software, the data was gated to exclude debris or aggregates. Experiments were performed in triplicate using cytochalasin D, which has been reported to arrest the cell cycle [35 (link)], as a positive control.

MCF-7 and BT20 cells were seeded in a 96-well plate (Corning, USA) at a density of 8,000 cells/well and were subsequently treated with 100 μM of DMDD for 24 h. The percentage of cells releasing cytochrome c from mitochondria was determined using the FlowCellect Cytochrome c Kit (Millipore, USA) according to the manufacturer's protocol. Briefly, cells were permeabilized, fixed, and stained with either anti-IgG1-FITC Isotype control or anti-Cytochrome c-FITC dye. Data was acquired and analyzed using a Guava flow cytometer and InCyte software (Millipore, USA).

Raw cells were seeded in 6‐well plates at a density of 4 × 10⁵. By using flow cytometry, the macrophage subpopulation markers CD16/32 (M1) and CD206 (M2) were used to assess different phenotypes. After each group of cells was cultured for 24 hours under different conditions, the cells were trypsinised for flow cytometry analysis. The Mouse CD16/32 PE and the Mouse CD206 Alexa 647 were incubated separately according to the manufacturer's instructions. Finally, they were analysed on a Guava flow cytometer (Millipore). Data were analysed using guavaSoft 3.1.1 software.

To simulate the inflammatory environment in the early stage of fracture, lipopolysaccharide (LPS, 1 μg/ml, Sigma) was added to the culture medium to activate RAW cells for 3 h. Next, conditioned medium from the O-N and O-EMF groups was used to culture RAW cells. The cells cultured in α-MEM were defined as the control group, and the inactive RAW cells were used as the blank group to be cultured in α-MEM. After 1 day of incubation, the expression levels of macrophage polarization-related genes [interleukin-6 (IL-6), interleukin-1β (IL-1), CD206, transforming growth factor-β1 (TGF-β), platelet-derived growth factor-B (PDGFB), VEGFA)] were detected by RT-qPCR.
Cell surface markers (CD86 and CD206) related to macrophage polarization were detected by flow cytometry. In detail, each group of cells treated with the above methods was collected. Then RAW cells were washed 3 times with PBS and resuspended in 1% bovine serum albumin (BSA) for 15 min at 4 °C to block nonspecific antigens. Next, APC-CD206 antibody (0.25 μg/test, Thermo Fisher Scientific) and PE-CD86 antibody (0.125 μg/test, Thermo Fisher Scientific) were incubated with the RAW cells for 15 min at ambient temperature. After washing twice with PBS, cells were resuspended in 100 μl of 1% BSA and analysed on a Guava flow cytometer (Millipore, USA). Data were analysed by Flowjo_V10 software.

RAW cells were seeded onto 6-well plates (5 × 10⁵ cells/well). Macrophage cell subpopulation markers CCR7 (M1) and CD206 (M2) were analyzed by flow cytometry to evaluate the different phenotypes. After 1 and 4 days of culture, cells were trypsinized, scraped from the plates, centrifuged, and resuspended in 1% bovine serum albumin (BSA) for 30 min at ambient temperature to block non-specific antigens. Then the cells were incubated with allophycocyanin (APC)-conjugated CCR7 and phycoerythrin (PE)-conjugated CD206 for 30 min at ambient temperature. The isotype controls used were APC-conjugated Armenian hamster IgG and PE-conjugated rat IgG2a,κ. All antibodies used for flow cytometry were purchased from eBioscience. After washing twice with PBS, cells were resuspended in 1% BSA and analyzed on a Guava flow cytometer (Millipore, USA). Data were analyzed using guavaSoft 3.1.1 software.

MCF-7 and BT20 cells were first synchronized in the G0 phase by culturing cells for 24 h in serum-free medium, and then treated with 100 μM DMDD for 24 h. Cell cycle analysis was performed using propidium iodide (PI) (Millipore, USA) staining as described [27 ]. Cells were analyzed using a Guava flow cytometer with InCyte software (Millipore, USA).