Sterile cell strainer

Following 6 days of cardiac differentiation, cultures were enzymatically digested by using collagenase A (6.67 mg/ml, Roche) and collagenase B (6.67 mg/ml, Roche) in differentiation media for 30 min. In order to obtain a uniform single cell suspension, cell clumps were digested 5 min with Accutase (Invitrogen). For FACS-sorting live Isl1-CPC, single cell suspension was filtered through a sterile cell strainer (BD biosciences). GFP- and GFP+ cell populations were sorted into 1∶1 FBS and differentiation media by using Mo-Flo FACS-sorter (Beckman Coulter) at cell sorter core facility at Yale University.
For FACS analysis of cardiomyocytes, single cell suspension was prepared as described above and fixed with 2% paraformaldehyde (PFA) in PBS. Next, fixed cells were blocked and permeabilized in PBST containing 10% Normal Goat Serum for 60 min. Samples were incubated with primary antibody for cTnT in PBST for 60 min at RT, followed by Alexa fluor-647 secondary antibody (Invitrogen) for mouse for 30 min at RT. The labelled cell population was analysed by using LSRII (BD biosciences) and Flow-Jo software (Treestar).

The interstitial cells were isolated from the testes of the all-treated groups of mice (3 weeks after injection). Testes were decapsulated and mechanically separated by few accurately aspirations through pipette tips and a 1-mL syringe containing phosphate-buffered saline [PBS, Beit HaEmek Biological Industries (Beit HaEmek, Israel]. The tubules were separated from interstitial tissue by gravity. The suspension of the single cells was filtered through a sterile cell strainer (70 µM; BD Biosciences) and washed with 5 mL PBS (centrifugation at 100× g for 5 min). After centrifugation, the media were removed and the pellet of the cells in the bottom of the tube was suspended in 1 mL of PBS.

The tubular cells were isolated from the testes of the BU-treated mice (10 days after BU injection). The testicular cell suspensions were obtained as described by Abu Elhija et al. [35 (link)]. Briefly, the testes were decapsulated and mechanically digested by multiple aspirations through pipette tips and a 50-mL syringe containing 20-mL phosphate-buffered saline (PBS). The tubules, which were settled by gravity, were subjected to enzymatic digestion (collagenase type V and DNAse). The suspension of the single cells was filtered through a sterile cell strainer (70 μM; BD Biosciences) and washed with 5 mL MEM (minimum essential media) (centrifugation at 100× g for 5 min). After centrifugation, the media were removed and the pellet of the cells in the bottom of the tube was suspended in 1 mL of fresh StemPro-34. The cells were suspended in StemPro-34 and were counted under phase-contrast microscopy in a Neubauer counting chamber.

Testes from seven-day-old and seven-week-old mice were surgically removed. Thereafter, mechanical followed by enzymatic disintegration of the tissue was performed. The enzymatic digestion solution was composed of collagenase 1 (mg/mL) and DNAse I (1 mg/mL). Both collagenase and DNase were dissolved in PBS, and isolated cells were filtered through a sterile cell strainer (70 µM; BD Biosciences; San Jose, CA, USA) and centrifuged for 10 min at 1500 RPM (300× g) [27 (link)] After centrifugation, cells were diluted in 1 mL of medium (StemPro Thermo Fisher Scientific, Waltham, MA, USA, with 10% KSR Thermo Fisher Scientific Gibco, Waltham, MA, USA). The total number of viable cells, using trypan blue staining, was counted under a light microscope. For DNA content analyses, the cells were fixed in a 4% paraformaldehyde (PFA) solution (Santa Cruz, CA, USA) for 15 min in ice [33 (link)].

The tubular cells were isolated from the testes of healthy, 7-day-old mice. The testicular cell suspensions were obtained as mentioned by Elhija et al., 2012 [21 ]. Shortly, tunica albuginea was removed and seminiferous tubules were mechanically digested and immersed in sterile phosphate-buffered saline (PBS) (Biological Industries, Beit HaEemek, Israel). After centrifugation testes were digested enzymatically (collagenase type V and DNAse) for 15 min at 37 °C. The cell suspension was filtered through a sterile cell strainer (70 µm; BD Biosciences) and washed with PBS. After centrifugation, the pellet of the cells was suspended in 1 mL of fresh StemPro-34 with 10% knock-out serum replacement (KSR) (Thermo Fisher Scientic, Waltham, MA, USA) and were counted under phase-contrast microscopy in hemocytometer.

Mice were euthanized using halothane followed by cardiac puncture to confirm death. Animals were then thoroughly perfused with ice-cold PBS, pH 7.4, for 5 mins. Heads were removed and skulls were stripped of all flesh. Surgical scissors were used to remove skull tops in a clockwise manner. The skulls were immediately placed in ice-cold RPMI media. Meninges were carefully removed from the interior aspect of skulls and surfaces of brains with forceps (21 (link), 22 (link)). Hippocampus was separated from the brain parenchyma using surgical forceps. Single cell suspension from meninges and hippocampus (HPC) was prepared by enzymatic digestion in RPMI containing 220 U/mg Collagenase IV (Gibco, Waltham, Massachusetts), 13 U/mg DNase I (Sigma, St. Louis, Missouri), and 5% iFCS (inactivated fetal calf serum) (Gibco) (digestion buffer) for 30 min at 37°C under constant rotation. The resulting suspension was mechanically squeezed through a 100 µM sterile cell strainer (Falcon) followed by centrifugation at 1200 rpm for 10 min at 4°C. Supernatant was discarded and the cells were resuspended in RPMI medium and checked for viability and cell number by trypan blue staining.