40 μm nylon filter

Ovaries were collected from 8dpp CD1 mice, cleared of the surrounding tissues and incubated in 1 mg/mL collagenase type I (Sigma-Aldrich) in αMEM for 1 hr at 37°C in agitation. Following collagenase removal, the ovaries were suspended in the cultured medium (see below) and disaggregated by gently pipetting. Cell suspension was filtered through a 40 μm nylon filter (BD Falcon) while the retained tissue fragments were further incubated in 0.25% trypsin (Sigma-Aldrich) for 15 min at 37°C in agitation, disaggregated and filtered as above. The cellular suspensions were then mixed and finally plated at 30.000 cells/cm². Culture was carried out in αMEM (Aurogene) supplemented with 10% FBS (Gibco), L-glutamine, penicillin-G and streptomycin, pyruvic acid, N-acetyl-L-cysteine (all from Sigma-Aldrich) and ITS liquid media supplement (Gibco), at 37°C in 5% CO₂ in 95% humidified incubator. The day after ( = T0 time point), 0.5 μM EPI, 10 μM CS or 10 μM PM was added to the culture medium of the treated groups. Where indicated, 200 mIU/mL LH was also added to the medium 1 hr before drugs.
Images were acquired under phase contrast microscope (Leitz Diavert) equipped with a DS-Fi1 camera (Nikon).

HL-60 cells (2 × 10⁶/mL) were treated with vehicle or tricetin (0, 20, 40, and 80 μM) for 24 h and then cells were collected and fixed by 70% ethanol. Next, cells were incubated with propidium iodide (PI) buffer (4 μg/mL PI, 0.5 mg/mL RNase A, and 1% Triton X-100 in phosphate-buffered saline (PBS)) for 30 min at 37 °C in the dark followed by filtration through a 40-μm nylon filter (Falcon, San Jose, CA, USA). The cell cycle distribution was analyzed for 10⁴ collected cells by a FACS Vantage flow cytometer that uses the Cellquest acquisition and analysis program (Becton-Dickinson FACS Calibur, San Jose, CA, USA). The proportion of nuclei in each phase of the cell cycle was determined, and apoptotic cells with hypodiploid DNA peak were catched in the sub-G₁ region.

Total ascites was collected when the mice were sacrificed. Erythrocytes in ascites were lysed using Red Blood Cells Lysis Buffer (Solarbio). Cells were washed using PBS with 1% FBS and passed through a 40 μm nylon filter (BD Biosciences, San Diego, CA, USA), then cells were Fc-blocked with anti-CD16/32, and stained with CD45-PE, CD11b-APC, and F4/80-APC/CY7 antibodies for detecting macrophages. MHC II-PERCP/Cy5.5 and CD206-FITC antibodies were used to differentiate M1- and M2-subtype macrophages. For detecting T cells and B cells, cells were stained with CD45-PE, CD3-FITC, CD4-APC, CD8-PE/CY7, and CD45R/B220-FITC antibodies. Cells were incubated with the antibodies for 30 min in the dark and washed twice using PBS with 1% FBS. LSRFORTESSA (BD Biosciences) was used to obtained the data, and FlowJo software (BD Biosciences) was used to analyze the data.

Muscle stromal cells (containing satellite cells) were isolated, as described previously, with modifications [28 (link), 29 (link)]. Briefly, portions of the back muscles of rats were minced under a dissection microscope and enzymatically digested with 0.2% Collagenase Type I solution (Worthington Biochemical, Lakewood, NJ) in DMEM at 37°C for 30 min, and 0.25% Trypsin-EDTA for 15 min, the cell suspension was filtered through a 40 μm nylon filter (BD Biosciences). The suspended cells were cultured in 6-well plates with DMEM supplemented with 10% FBS (Invitrogen), and 1.0% penicillin-streptomycin for 24 hours. The isolated cells were then fixed and perforated with fixation and permeabilization solution (BD Biosciences). Then, the isolated cells were incubated with FITC labeled Anti-PAX7 antibody (Abcam) and underwent flow cytometry analysis (FACS Calibur™, BD Biosciences). The PAX7 positive cells were quantified.

Single-cell suspensions from naïve WT, Rag1^−/−, and NRG mouse spleens and lymph nodes (LN; cervical) were prepared. Tissues were homogenized and strained through a 40-μm nylon filter (BD Biosciences, Germany). Homogenates were rinsed with washing medium (Dulbecco’s Modified Eagle’s Medium, DMEM, Invitrogen, USA) containing 1% FBS (ScienCell, USA), 1% glutamine (Gibco Life Technologies, USA), and 1% antibiotics (Sigma-Aldrich, USA) and shortly resuspended in erythrocyte lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA; pH 7.3).
Brain tissues were cut into pieces, homogenized in phosphate-buffered saline, layered on a density gradient using Lymphoprep™ (Fresenius, Germany), and separated by centrifugation for 16 min at 500 g. After isolating cells, they were washed and resuspended in the respective staining buffer. To quantify numbers of cells isolated from the central nervous system, beads (Beckman Coulter, USA) were added.