Cell strainer tubes

Frozen PBMCs were rapidly thawed at 37°C and gently resuspended by serial additions of Lymphocyte medium to a final volume of 10 mL. Cells were centrifuged at 300 x g for 6 minutes and resuspended in 1 mL of Lymphocyte medium and 10 U/mL DNase I (New England Biolab, Cat: M0303). PBMCs (3.0×10⁶ cells) were stained by incubation with the following panel at 4°C for 20 minutes: FcR blocking reagent (Miltenyi Biotec, 1:50), anti-αβTCR-PE/Cy7 (BioLegend, Clone: IP26, Cat: 306720, 1:100), anti-CD3-BV421 (BioLegend, Clone: UCHT1, Cat: 300434, 1:100), anti-CD4-redFluor 710 (Tonbo Biosciences, Clone: OKT4, Cat: 80–0048, 1:100), anti-CD8-PerCP/Cy5.5 (BioLegend, Clone: RPA-T8, Cat: 301032, 1:100), anti-CD14-APC/Cy7 (BioLegend, Clone: HCD14, Cat: 325620, 1:100), anti-CD16-APC (Tonbo Biosciences, Clone: 3G8, Cat: 20–0166, 1:100), anti-CD19-Super Bright 645 (eBioscience, Clone: SJ25C1, Cat: 64–0198-42, 1:100), and anti-CD56-Alexa Fluor 488 (BD Biosciences, Clone: B159, Cat: 557699, 1:100) antibodies. Cells were then spiked with 7-AAD (Tonbo Biosciences, 1:200) and incubated at 4°C for 10 minutes. Cells were washed once, resuspended in 200 μL of FACS buffer, and filtered in Cell Strainer tubes (Corning, Cat: 352235). Cells were sorted with a BD FACS Aria (BD Biosciences).

Long bones were isolated and marrow plugs flushed as previously described (29 (link)). Bones were then broken into small pieces with surgical scissors and incubated in Collagenase II (Sigma Aldrich) buffer (DMEM, 2% FBS, 1% Penicillin/Streptomycin, 2mg/mL Collagenase II) for one hour at 37°C and 200 RPM. The solution was then filtered through a 70μm filter, and then washed with Hemolytic Buffer. Hemolyzed cells were then filtered into Cell-Strainer tubes (Corning, Corning, NY, USA) and resuspended in FACS Wash (DPBS, 2%FBS), stained with antibodies, and then sorted/analyzed using flow cytometry. In vitro cultured cells were propagated in MSC media (MEMα, 15% FBS, 1X penicillin/streptomycin) at 37°C, 5% CO₂, and >95% humidity.