Shandon cytospin 4

Assessment of mitochondrial morphology was carried out as previously described [15 (link)]. Cells were seeded in 6-well plates, trypsinized, and washed with phosphate buffered saline and centrifuged (900 x g, 1 min). 100μl of the cell suspension was centrifuged (450 x g, 4 min; Shandon Cytospin 4, Thermo Fisher Scientific) using cytological funnels together with silane-coated glass slides [29 (link)]. For immunostaining, cells were fixed in 4% paraformaldehyde (1 h, room temperature (RT)) and blocked with 3 % bovine serum albumin. Mitochondria were incubated with anti-Tom20 antibody and appropriate secondary antibody. Confocal images were obtained on Zeiss LM510 inverted microscope (Carl Zeiss, Jena, Germany) with appropriate argon lasers (488nm) and 40X objective. Mitochondrial phenotype of each cell was categorized as tubular or fragmented, as previously described [29 (link)]. A cell with “fragmented mitochondria” was defined as the cell with 8 or more individual mitochondrial fragments that were each 3 μm in length across the longest axis. At least 300 cells were analyzed per experimental group.

Cells were treated with a hypotonic solution, 75 mM KCl for 15 min at 37°C then spun onto coverslip (ZEISS) coated with poly-L-lysine (SIGMA) by cytocentrifugation (Shandon Cytospin 4, Thermo) at 1,300 rpm for 10 min. Then, cells were fixed with 2% PFA (para-formaldehyde) in PBS for 15 min, or with iced-cold 100% methanol at -20°C for 15 min, and permeabilized by 0.2% Triton X-100 in PBS for 10 min. Cells were blocked with 3% BSA in PBS for 1 hr then primary and secondary antibody reactions were performed under the same condition, at room temperature for 1 hr, respectively. Samples were mounted in Vectorshield mounting medium (Vector Laboratories). DNA was counterstained by Hoechst 33342 or DAPI. Samples were visualized by fluorescence microscopy, Axioplan 2 (ZEISS) or DeltaVision (GE Healthcare).

To induce mitotic arrest, approximately 1 × 10⁵ cells were treated with 0.5 μg/mL Colcemid Solution (Gibco/ThermoFisher) for 2 h in a 28°C heat block. Cells were then spun for 5 min at 600x g at RT to pellet and resuspended in hypotonic solution (500 mL of 0.5% sodium citrate). Cells were incubated in hypotonic solution for 8 min at RT. 100 μL of the cell suspension was then placed in a cytofunnel and spun at 1200 rpm for 5 min with high acceleration using a cytocentrifuge (Shandon Cytospin 4; ThermoFisher). For FISH, slides were then fixed in cold 3:1 methanol: acetic acid for 10 min and washed 3X 5 min in PBS-T (PBS with 0.1% Triton X-100). FISH was performed as described above for meiotic squashes.

Sorted CD71⁺Ter119⁺ fetal liver erythroblasts from E11.5 embryos were attached to a slide glass using Shandon Cytospin 4 (Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde phosphate buffer solution (Nacalai Tesque) for 15 min, permeabilized with 0.5% Triton X‐100 and 0.1% gelatin in PBS for 10 min and blocked with 0.1% gelatin and 2% goat‐serum (#143-06561; FUJIFILM Wako Pure Chemical) in PBS for 30 min at room temperature. Then cells were incubated with primary antibody against γ-H2AX (Ser139, #2577, Cell Signaling Technology) or GATA-1 (#3535, Cell Signaling Technology), followed by the incubation with secondary antibody conjugated with Alexa Fluor 568 (Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific). After washing, samples were mounted using ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and air-dried. Images were acquired using TCS SPE (Leica) and analyzed using ImageJ (NIH). The presence of micronuclei and γ-H2AX was determined manually by two independent investigators.

The lungs were lavaged with 4 × 0.5 ml of PBS. The BALF was centrifuged 207 g at 4°C for 10 minutes, and the supernatant was collected and stored at -80°C for further analysis. The cells were resuspended in 1 ml of PBS and the total cell numbers were counted with a hemocytometer. Cytospin samples were prepared by centrifuging the suspensions at 350 rpm for 10 minutes using Shandon Cytospin 4 (Thermo scientific, Cheshire, UK) and cell differentials were determined by counting at least 300 leukocytes on Giemsa stain according to manufacturer’s instructions.