Falcon cell strainer

Mfsd7c^fl/fl and Mfsd7c^−/− (Mfsd7c^fl/flLysMCre^+/+) C57BL/6 J mice were euthanized using CO₂ asphyxiation. Femoral bones were removed and cleaned and the bone marrow was flushed out using 5 mL of cold DMEM media. Bone marrow cells were collected by centrifugation (1200×g for 5 min at 4 °C) and resuspended in 4 mL of ACK lysis buffer. After incubation at room temperature for 5 min, ACK buffer was neutralized by the addition of 11 mL of cold DMEM media and cell suspension was centrifuged at 1200×g for 5 min at 4 °C. Cells were resuspended in DMEM media containing 10% FBS, 20% L929-conditioned media, 2 mM L-glutamine, 2 mM pyruvate, non-essential amino acids (100 μM each), 0.55 mM 2-mercaptoethanol, penicillin/streptomycin), passed through a 40 μm Falcon cell strainer (VWR) to remove large aggregates, and counted. Bone marrow cells were seeded in 10 cm non tissue-culture treated plates at 10 million cells per plate in 10 mL of BMDM media. Two days later, additional 10 mL of BMDM media was added. On day 4 and day 6, old media was removed and 10 mL of fresh media was added. On day 7, fully differentiated BMDM were lifted in phosphate-buffered saline supplemented with 10 mM EDTA for 5 min at 37 °C, centrifuged and resuspended in BMDM media to a proper density for further experimentation.

Single cell suspensions of lymphocytes from spleens and lymph nodes were obtained from mice by pressing spleens or lymph nodes through a 70 μm Falcon Cell Strainer (VWR, Mississauga, ON) into RPMI 1640 medium (GIBCO). Cells were then centrifuged (300 x g, 10 min, 4°C), and erythrocytes were lysed using Ammonium-Chloride-Potassium (ACK) red cell lysis buffer. The resulting live (trypan blue-negative) splenocytes and lymphocytes were counted manually (microscope slide) and stained directly or cultured for further assessment.

For each sample, 400 male adult flies were anesthetized, frozen in liquid nitrogen and stored at −80 °C. For nuclei isolation, frozen flies were submitted to five rounds of vortexing and cooling in liquid nitrogen and heads were separated from thoracicoabdominal segments, wings and legs using two different-sized pre-chilled sieves (Hogentogler, 710 μm and 425 μm pore sizes). Separated heads were transferred into 1 mL of Nuclei Extraction Buffer (15 mM Hepes [Na+], pH 7.5, 10 mM KCl, 5 mM MgCl₂) in a Dounce homogenizer and incubated on ice for 5 min. Nuclei were extracted by using five strokes with a loose pestle, followed by an incubation on ice for 5 min and subsequent 5 strokes with a loose pestle. Head homogenate was filtered through a 40 μm Falcon cell strainer (VWR, cat # 21008-949) and immunoprecipitated with 10 μg of GFP antibody (Roche, cat # 11814460001) as previously described [29 ] with the following modifications: The salt concentration in the PBS wash buffer was supplemented to a final concentration of 300 mM NaCl, and five washes were conducted. Detailed protocols are provided via PURR.

Draining lymph nodes and spleens were obtained from tumor-bearing mice, lymph nodes and spleens were also isolated from naïve mice. Lymph nodes were pressed through a 40 μm Falcon Cell Strainer (VWR, Mississauga, ON) into RPMI 1640 medium (Gibco) and cells were counted and centrifuged for 5 min at 1,500 rpm/4°C. Spleen cells were further isolated using ACK Lysing Buffer (Lonza) to lyse red blood cells.