Maintenance of Drosophila S2 cells and CO2 exposure

Maintenance of Drosophila S2 Cells and CO₂ Exposure

Cell culture conditions
Drosophila S2 cells were maintained at room temperature and protected from light in Schneider’s Insect Medium supplemented with 10% fetal bovine serum (FBS; Valley Biomedical) and 0.2% Penicillin-Streptomycin (Gibco). Cells were cultured under standard non-CO₂ incubator conditions.

Plate preparation for cell attachment
Six-well plates were pre-treated to promote S2 cell attachment. Wells were incubated with 1 N HCl for 1 h at room temperature, rinsed three times with sterile water, and then coated with 0.5 mg/mL Concanavalin A (ConA; Sigma) for 1 h. After incubation, plates were washed once with sterile water and allowed to dry in a sterile environment. S2 cells were seeded at 2.0 × 10⁶ cells per well and allowed to attach for 1 h before experimental manipulation.

Preparation of CO₂-equilibrated medium
For CO₂ exposure experiments, the culture medium was prepared using Schneider’s Insect Medium, Tris base, and MOPS base at a ratio of 4:0.25:0.25 (v/v). This mixture contained 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. To adjust buffering capacity, the initial pH was titrated with Tris and MOPS to achieve a final pH of 7.2 when equilibrated at a CO₂ partial pressure of ~120 mmHg.

The prepared medium was equilibrated overnight at room temperature, protected from light, in a C-Chamber (BioSpherix). CO₂ concentration within the chamber was controlled using a PRO CO₂ Controller (BioSpherix). After equilibration, pH, pCO₂, and pO₂ levels were verified using a Stat Profile pHOx blood gas analyzer (Nova Biomedical).

CO₂ exposure
At the start of each experiment, the culture medium in each well was replaced with the CO₂-equilibrated medium. Cells were incubated at room temperature, protected from light, for the duration of CO₂ exposure.

Reference

1. Shigemura M et al. Elevated CO₂ regulates the Wnt signaling pathway in mammals, Drosophila melanogaster and Caenorhabditis elegans. Sci Rep 2019 Dec 3;9(1):18251.