Moflo flow cytometer

The cells were harvested and washed with PBS then re-suspended in cold 1% formaldehyde in PBS solution for 15 min at 4°C. The cells were washed twice in PBS and re-suspended in ice-cold 70% ethanol and stored at −20°C for 2 h prior to analysis. Prior to fluorescence measurement the cells were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). The intensity of blue fluorescence emission of DAPI stained DNA, excited with the UV laser (355 nm) was measured, recorded and analyzed on a MoFlo flow cytometer (Beckman Coulter Life Sciences, Indianapolis IN, USA) using Kaluza fluorescence intensity analysis software (48 (link)). Experiments were repeated and representative data are presented.

For CIK cells harvested on days 7, 14, 21 and 28 were washed twice with washing buffer (PBS buffer containing 0.5% BSA), blocked with blocking buffer (PBS containing 2% BSA) for 10 min at 4°C and washed twice again. Then the washed CIK cells were stained with 5 µl of the following monoclonal antibodies in 100 µl blocking buffer (PBS containing 1% BSA) for 30 min at 4°C, washed twice, resuspended in 100 µl PBS buffer and analyzed via flow cytometry. The following antibodies were used and diluted 1:20 in blocking buffer (PBS containing 1% BSA): CD3-FITC (cat. no. 11-0036-42), CD56-APC (cat. no. 17-0567-42), CCR4-PE (cat. no. 12-1949-41), CCR5-PE (cat. no. 12-1956-41), CCR7-PE (cat. no. 12-1979-42), CXCR3-PE (cat. no. 12-1839-42) and CXCR4-PE (cat. no. 12-9999-41) antibodies, as well as the corresponding isotype controls IgG2a-PE (cat. no. 12-4321-80), IgG1a-PE (cat. no. 12-4714-82), IgG1a-FITC (cat. no. 11-4714-81) and IgG1a-APC (cat. no. 17-4714-82). All antibodies were purchased from eBioscience; Thermo Fisher Scientific, Inc. Data were obtained using a MoFlo flow cytometer (BeckmanCoulter, Inc.) and analyzed using Summit version 5.2 (Beckman Coulter, Inc.) and FlowJo version 10 software (Becton, Dickinson and Company).

Skeletal muscles from both fore‐limbs and hind limbs were dissected out and digested with 0.2% collagenase type II (Worthington Biochemical CorporaAon, Lakewood, Washington, www.worthington-biochem.com) for 1 hour at 37°C. Then, the digested tissue was filtered through 100 µm‐ and 40 µm‐cell strainers (BD Biosciences). The filtered mononuclear cells were stained with phycoerythrin (PE)‐conjugated anti‐CD31 (BD Biosciences), PE‐conjugated anti‐CD45 (BD Biosciences), FITC‐conjugated anti‐Sca‐1 (BD Biosciences), and biotinylated SM/C‐2.6 antibodies 24 on ice for 30 minutes. After washing, streptavidin‐allophycocyanin (BD Biosciences) was added to the cells labeled with biotinylated SM/C‐2.6 antibody and incubated on ice for 30 minutes. All the cells were resuspended in HBSS (−) and propidium iodide. Cell sorting was performed using MoFlo flow cytometer (BeckMan, Brea, California, www.beckmancoulter.com), and CD31⁻, CD45⁻, Sca‐1⁻, and SM/C‐2.6⁺ cells were collected as satellite cells 24. Percentage of satellite cells in the total mononuclear cells, except for CD31‐positive endothelial cells and CD45‐positive lymphocytes/leukocytes, was calculated for analyzing satellite cell population.

FAM46C gene was knocked out from RPMI‐8226 cells using CRISPR‐Cas9 system. The sgRNA was cloned into a pSpCas9(BB)‐2A‐mCherry(px458) vector from Prof. Jichang Wang's lab at Zhongshan School of Medicine, Sun Yat‐sen University (Guangzhou, Guangdong, China). The recombinant plasmid was amplified in E. coli DH5α (AlpaLife, Shenzhen, Guangdong, China) and transfected into RPMI‐8226 cells with Lipofectamine^TM 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. The cells were transferred into 12‐well plates and cultured for 48 hours. RPMI‐8226 cells carrying recombinant pSpCas9(BB)‐2A‐mCherry(px458)‐gRNA^FAM46C plasmid were sorted using a fluorescent activity cell sorters (FACS) MoFlo flow cytometer (Beckman, LA, CA, USA) at a wavelength of 561 nm and allocated to 96‐well plates. After 2‐month culturing of these single clones, knockout of FAM46C was examined by Sanger sequencing and Western blotting.