Synergy sy3200

Testes from adult C57BL/6 (JAX®mice, stock no. 000664) and transgenic mice were excised and the tunica albuginea was removed. Briefly, seminiferous tubules were transferred to 10ml of digestion buffer1 (comprised of Advanced DMEM:F12 media (Life Technologies), 200 μg/ml Collagenase IA (Sigma), and 400 units/ml DNaseI (Worthington Biochemical Corp.)). Tubules were dispersed by gently shaking by hand, and allowed to settle for 1 min at room temperature. Tubules were then transferred to digestion buffer 2 (200 μg/ml trypsin (Invitrogen) and 400 units/ml DNaseI (Worthington Biochemical Crop) dissolved in Advanced DMEM:F12 media) and dissociated at 35°C / 215 rpm for 5 min each and quenched with the addition of fetal bovine serum (FBS). Cells were filtered through a 100 μm strainer, washed in Phosphate-buffered saline (PBS), pelleted at 600g for 3min, and re-suspended in MACS buffer containing 0.5% BSA (MACS buffer; Miltenyi Biotec). For interstitial cell enrichment, testes were dissociated in digestion buffer 1 (described above) for 5min at 35°C / 150 rpm, the cells were dislodged by a gentle hand shake, supernatant quenched with FBS (tubules were discarded), and used directly for Drop-Seq. For all Drop-seq experiments, live single-cell suspensions were collected by flow cytometry using FACSARIA II/III (BD Biosciences) and Synergy SY3200 (Sony) cell sorters.

For flow cytometry experiments, the SVF suspension was isolated and blocked as mentioned above. Cells were then stained with specific cocktails of antibodies for selected clusters, Cluster 1, CD45-PECy7, CD11b-Alexa Fluor 488, CD9-APC, CD11c-PE; Cluster 2: CD45-FITC, CD11b-PE, CD206-APC, LYVE-1-PECy7; Cluster 5: CD45-FITC, CD11b-Alexa Fluor 594, Ly6a-PE, and CD34-PE/Cy7. For intracellular staining of collagen, cells were preincubated with Intracellular Staining Permeabilization Buffer (BioLegend) followed by blocking in 2.5% goat serum diluted in phosphate-buffered saline (PBS). Cells were incubated with primary rabbit anti-mouse COL1A1 antibody (Antibodies-online.com), followed by wash and secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488. Finally, CD45-APC, CD11b-PE/Cy7, and Ly6a-PE were added. Data were collected by flow cytometry analyzer (BDX-20, BD Bioscience). For the in vitro study, cells were stained with CD45-APC, CD11b-AlexaFluor 488, Ly6a-PE, and sorted (Sony SY3200 “Synergy”). Antibody catalog numbers and dilutions are presented in Supplementary Table S2.

For scRNA-seq, flow cytometry analysis, and flow cytometry sorting, mouse Epi/VAT biopsies were minced and digested in 4–6 mg/mL collagenase I (Sigma-Aldrich), 4–6 mg/mL collagenase II (Roche), and 1% BSA (Sigma-Aldrich) in Hanks Balanced Salt Solution (Sigma-Aldrich) at 37°C for 45 minutes with agitation (180 rpm), then, strained through a 70-μm strainer in PBS with 0.1% BSA. The cell suspension was centrifuged at 300 ×g for 5 minutes, the supernatant removed, and the cell pellet resus-pended in PBS with 0.1% BSA. Red blood cell lysis was performed (BioLegend), followed by wash and centrifugation (300 ×g, 5 minutes), resuspension in PBS, and straining through a 40 μm filter. For scRNA-seq, the SVF suspension was blocked in the anti-mouse CD16/32 antibody (BioLegend) for 30 minutes at 4°C followed by antibody incubation with CD11b-Alexa Fluor 488 and CD45-APC (BioLegend) for 30 at 4°C. Cells were then washed twice with PBS and sorted using fluorescence-activated cell sorting (FACS) (SY3200 Synergy, Sony Biotechnology).

pTUB-ROP21YFP-CAT was used as a template in Q5 Mutagenesis (NEB) reactions to engineer point mutations at the two potential ASP5 processing sites and these were confirmed by sequencing. Fifteen micrograms of each plasmid were used to transfect RH T. gondii that were seeded into T25 flasks of HFFs and incubated for 36 hours. Monolayers were dissociated using trypsin (Life Technologies) and suspended in 3% FCS PBS with additional 5mM EDTA, filtered using a 70 μm cell strainer (Falcon). Samples were analyzed using a Sony SY3200 “Synergy” and YFP positive events were sorted into 500 μl D10. These were recovered by centrifugation at 1000 × g for 10 min, and then 15,000 cells per sample were analyzed by western blotting as above, with the exception that YFP was detected by α-GFP mouse monoclonal JL8 (Living Colors, Clontech), loading control was rabbit anti-T. gondii aldolase (TgAld) (Starnes et al., 2006 (link)).