Nylon cell strainer

Cell lines were plated and cultured in 6-well plates for 48 h prior to collection or differentiation treatment. Cultures (1.0-2.0 × 10⁶ cells per well) were dissociated and washed in PBS. Cultures were then resuspended and treated with LIVE/DEAD Near-IR dye (ThermoFisher) according to manufacturer’s recommendations. After treatment, cells were washed in PBS, filtered through a 40 μm nylon cell strainer (Corning), and resuspended in 1 mL sorting media (DMEM, 1% FBS, Penicillin/Streptomycin). Treated cultures were then sorted with a BD FACSAria II flow cytometer using 639 nm excitation per biological replicate using the services of the Duke Flow Cytometry shared resource facility at the Duke Human Vaccine Institute (DHVI). Flow cytometry sorting data was imported, parsed, and analyzed using the flowCore package (R 3.6.0, RStudio 1.4.1717). Cell death was defined as any single cell event with IR emission intensity greater than 40x the IR intensity median per sorting experiment, with the percentage of dead cells reported for each biological replicate. To calculate cell death across biological replicates in cultured, undifferentiated, and differentiated SH-SY5Y cell lines, we subsampled 100,000 sorting events from each set of isogenic conditions. Plots were generated and statistical significance analyses were performed using R (3.6.0) in the RStudio (1.4.1717) software suite.

Cancellous-bone samples were weighed and then minced into 1–2 mm³ pieces with a sharp osteotome. Bone-derived cells were collected in two fractions as previously described [54 (link)]. The loosely adherent cells released by mincing and mechanical agitation were collected and defined as the MS fraction. Cells that remained adherent to the trabecular surface were then dissociated by enzymatic digestion with 111U/mL collagenase type-I and 24U/mL dispase (Worthington Biochemical, NJ) for 90 minutes at 37 °C. This fraction of cells was defined as the TS fraction. AT-samples were minced with a surgical scalpel and cells were released by enzymatic digestion with 111U/mL collagenase type-I and 24U/mL dispase for 90 minutes at 37 °C. Cells from each source were filtered through a 70 µm nylon cell strainer (Corning, NY) to separate tissue debris and to prepare suspensions of mostly single cells. Cells were centrifuged at 300×g for 5 minutes. The pellet was re-suspended in medium containing: alpha-minimum essential medium (αMEM), fetal-bovine serum (FBS) 16.5% (Atlanta Biologicals, GA), L-glutamine 2–4 mM, 1U/mL penicillin, and 0.1 mg/mL streptomycin (Sigma, MO). Cell number was determined using a hemocytometer. Cell concentration ([Cell]) (cells per gram of tissue) from MS, TS, and AT were calculated.

Epithelial cells including IELs and LP cells were isolated as previously described²⁶ using EDTA containing calcium-free media and collagenase VIII, respectively. For the analysis of the NK receptors by flow cytometry, cytotoxic molecules on IELs by flow cytometry and qPCR, and for the analysis of IFN-γ on LP cells, a cell purification step using 40% Percoll (GE Healthcare) was used to enrich lymphocyte cell populations. Briefly, PBS-washed epithelial and LP cells were resuspended in 10ml 40% Percoll solution then centrifuged for 30 minutes at 3000 x g. After removal of the supernatant, cells were washed in PBS and counted. Mesenteric lymph nodes were dissected, made into a single cell suspension by mechanical disruption and passed through a 70μm nylon cell strainer (Corning).