40 μm cell strainer

Fresh bovine nasoseptal cartilage biopsies were placed into sterile DMEM (Invitrogen, UK) and minced it into 1 mm³ pieces prior to sequential pronase (Roche, 0.4%, 1 hour at 37 °C) and collagenase (Sigma, 0.2% for 10 hr at 37 °C^{7 (link)}, digest with gentle agitation. The mixture was filtered through 40 μm cell strainer (VWR), centrifuged (500 rcf for 5 minutes) to remove enzyme mixture, re-suspended in culture media (containing DMEM supplemented with Penicillin 10000 mg/ml, Streptomycin 10000 U/ml, 0.1 mM ascorbic acid, 0.5 mg/ml L-glucose, 100 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine and 10% fetal bovine serum (FBS)) and seeded at 2700 cells/cm² for cell expansion^{45 (link),46 (link)}. Once > 70% confluent, cells were incubated in 0.05% trypsin-EDTA (Thermofisher) for 5–10 minutes at 37 °C for further expansion.

Spleens were mechanically dissociated in cell culture media. To obtain non-adherent peritoneal cells, peritoneal cavities were washed with DPBS as previously described.¹³ After sacrifice, the peritoneal cavity was injected with sterile DPBS, then massaged. The wash was collected and centrifuged at 300×g to collect cells. Red blood cells from the ascites were lysed using a buffer of ice-cold 155 mM NH₄Cl, 12 mM NaHCO₃, and 0.1 mM EDTA.
Tumours were dissociated using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany), then incubated at 5% CO₂ and 37 °C in RPMI-1640 with 1% l-glutamine, 1% pen-strep, 10% FBS, and 20 μg/ml Liberase (Roche, Basel, Switzerland) for 40 m. Samples from each tissue were passed through 40 μM cell strainer (VWR, Radnor PA, USA).

All tissue was dissected into 30 mg sections from DLF white matter with a scalpel blade and stored at − 80° C. On the day of single-nucleus encapsulation, tissue was placed in a 7 mL dounce with ice cold buffer (0.5 M sucrose, 2 M KCl, 1 M MgCl2, 1 M Tricine-KOH pH 7.8, spermine, spermidine, DTT, RNasin, H2O) and dounced with a tight pestle ten times to mechanically dissociate and homogenize the tissue. To further dissociate the tissue, 5% IGEPAL-CA630 was added to the homogenate and dounced 5 times. The homogenate was then strained through a 40 μm cell strainer (VWR) on ice. 5 mL of 50% iodixanol was added to the homogenate and mixed. In a 13 mL ultraclear ultracentrifuge tube, 1 mL of 40% iodixanol was added with 1 mL of 30% iodixanol carefully layered on top, followed by 10 mL of the tissue homogenate. The tissue was then ultracentrifuged in a swing-bucket rotor at 10,000xg for 18 min. Following ultracentrifugation, the 35% iodixanol layer was collected and the nuclei were counted. Nuclei were diluted with 30% iodixanol to 90,000 nuclei/mL for inDrops chip loading. Nuclei were isolated for inDrops using a previously described method [24 (link)].

Small luteal cells were isolated by a modified enzymatic method (14 (link)). CL were placed into a Petri Dish (60 × 10 mm, Thermo Fisher Scientific, Dreieich, Germany) containing medium I (HAM's F12 and MEM Eagle Medium 1:1 supplemented with 0.055 mg/mL gentamicin and 5% FBS), chopped into small pieces and transferred on a 40 μm cell strainer (VWR International, Dresden, Germany), which was placed into another Petri Dish. Pieces were covered with medium I supplemented with 0.1% collagenase (types I and II; SERVA Electrophoresis GmbH, Heidelberg, Germany) and 0.005% DNAse I and digested on the strainer for 55 min at 39°C. Thereafter, pieces were gently smashed through the strainer and the obtained suspension was agitated by pipetting and transferred to a glass tube for centrifugation (7 min at 1,000 × g). The cell pellet was resuspended in 2 mL fresh medium I and placed on 40% percoll solution (with DPBS) in a glass tube. After centrifugation for 7 min at 1,000 × g, cells were collected from the interphase between medium I and percoll solution, transferred to 1.5 mL reaction tubes and centrifuged at 500 × g for 4 min. The obtained cell pellet was resuspended in fresh medium I, the cell concentration was determined and set to 200,000 cells per mL (30,000 per 150 μL).

Thawed human ovarian cortical tissue was chopped using scalpels into pieces of ∼0.3 mm³ and enzymatically digested in DMEM/F12 (Thermo Fisher Scientific) containing 5% FBS, 1 mg mL⁻¹ collagenase IA (Sigma-Aldrich), 50 μg mL⁻¹ Liberase and 1000 U DNase I (Roche, Sigma-Aldrich) in a shaking 37 °C water bath for max. 50 min. Digestion was stopped with medium containing 10% FBS and cell suspension was centrifuged for 7 min on 300 × g. Cells were resuspended in DPBS, 2% FBS and passed through a 40 μm cell strainer (VWR, USA), counted and used for subsequent experiments.

Small intestine (SI) and colons were washed in HBSS with 2% FCS, following Peyer patch excision. Intestines were opened longitudinally and cut into 0.5 cm segments and incubated in HBSS with 2 mM EDTA (Sigma-Aldrich) at 37°C, shaking for 20 min to remove the epithelial cell layer and mucus. The SI lamina propria (LP) was digested with 1 mg ml⁻¹ collagenase VIII (Sigma-Aldrich) for 20 min, whereas colonic LP was digested with 0.85 mg ml⁻¹ of collagenase V (Sigma-Aldrich), 1.25 mg ml⁻¹ collagenase D (Roche, Penzberg, Germany), 1 mg ml⁻¹ dispase (Life Technologies, Paisley, U.K.), and 30 mg ml⁻¹ DNase (Roche) for 40 min. Cells were then passed through a 40-μm cell strainer (VWR, Radnor, PA) and stained for flow cytometry. LNs were separated and incubated with 0.4 Wunsch units ml⁻¹ Liberase TM and 50 mg ml⁻¹ of DNase (Roche) for 45 min at 37°C while shaking. Single-cell suspensions were passed through a 40-μm cell strainer and stained for flow cytometry. Lymph from thoracic duct cannulation was filtered through a 100-μm mesh, washed with HBSS EDTA, and spun at 400 × g for 5 min. Next, the cellular pellet was separated from the supernatant, and this was spun once more, this time at 6500 × g for 5 min to pellet and collect cell-independent bacteria.