Beckman moflo astrios eq

For flow cytometry analysis and isolation of hematopoietic stem and progenitor cells, cells were stained with relevant antibodies^{51 (link)} in PBS with 2% fetal bovine serum for 30−45 min on ice. Antibody clones that were used: Sca-1-PE-Cy7, c-Kit-APC, CD150-PE, CD48-BV421, CD45.1-FITC, CD45.2 PE-Cy5, Gr-1-PE-Cy5, Mac1-PE-Cy5, IgM-PE-Cy5, CD3-PE-Cy5, CD4- PE-Cy5, CD8-PE-Cy5, CD45R-PE-Cy5 and Ter-119-PE-Cy5. Detailed antibody information is summarized in Supplementary Table S6. HSPCs were stained with a lineage antibody cocktail (Gr-1, Mac1, CD3, CD4, CD8, CD45R, TER119 and B220), Sca-1, c-Kit, CD150 and CD48. Cell types were defined as followed: LSK compartment (Lin⁻Sca-1⁺c-Kit⁺), LT-HSC (LSK CD150⁺CD48⁻), ST-HSC (LSK CD150⁻CD48⁻), MPP2 (LSK CD150⁺CD48⁺) and MPP3/4 (LSK CD150⁻CD48⁺). B cells (CD45.2⁺Mac1⁻Gr-1⁺B220⁺), T cells (CD45.2⁺Mac1⁻Gr-1⁺CD3⁺) and myeloid cells (CD45.2⁺Mac1⁺Gr-1⁻). Samples were analyzed on a flow cytometer (CytoFLEX LX, Beckman). For sorting HSCs, lineage antibody cocktail-conjugated paramagnetic microbeads and MACS separation columns (Miltenyi Biotec) were used to enrich Lin⁻ cells before sorting. Stained cells were re-suspended in PBS with 2% FBS and sorted directly using the Beckman moflo Astrios EQ (Beckman). Flow cytometry data were analyzed by FlowJo (BD) software.

Mouse synovial tissues were isolated from knee joints, digested with 1 mg/ml type I collagenase in HBSS, and incubated at 37 °C and 5% CO₂ in a humidified atmosphere for 30–45 min. Disaggregated tissue elements were passed through a 70 µm cell strainer. Synovial macrophages (CD45⁺, CD11b⁺, F4/80⁺)^{50 (link)} were sorted on a BECKMAN Moflo Astrios EQ (BECKMAN Coulter). Next, total RNA was isolated according to the manufacturer’s instructions. Synthesis of cDNA and qRT-PCR was performed as previously described in vitro analysis.

The production of lentivirus for MC38‐Coro1a or MC38‐Coro1a‐K233R overexpression was carried out as follows. HEK293 cells were seeded into one 10 cm dish. The following day, the cells were transfected with 5 μg of plvx‐mCherry‐Coro1a‐Flag or 5 μg of plvx‐mCherry‐Coro1a‐K233R‐Flag, 3.75 μg of psPAX2 (gag, pol) and 1.25 μg of pMD2.G using 20 μl of JetPEI. Viral supernatants were collected 48 h after transfection and filtered through 0.45 μm PVDF filters. For initial lentiviral transduction, MC38 cells were infected with the appropriate virus in six‐well plates in the presence of 10 μg ml⁻¹ polybrene and centrifuged at 1500 × g for 2 h at 32°C. After 24 h, single mCherry⁺ cells were sorted into 96‐well plates with a Beckman MoFlo Astrios EQ (Beckman). The levels of endogenous and overexpressed proteins were then verified by WB.
For Coro1a knockout, HEK293 cells were seeded into 12‐well plates. The following day, the cells were cotransfected with plv5‐Cas9‐Blast (Merck) and U6‐gRNA: hPGK‐puro‐2A‐tBFP (Merck) including a specific gRNA sequence listed in Table S1. After 24 h, single BFP⁺ cells were sorted into 96‐well plates with a Beckman MoFlo Astrios EQ. The levels of endogenous and overexpressed proteins were then verified by WB.