Gibco cell culture media

MCF-7 cells (passage number < 10) were cultured in T-75 flask containing 10 mL RPMI1640 medium (Gibco^TM cell culture media, Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (iFBS) and 1% of penicillin/streptomycin (Gibco^TM, Fisher Scientific). The cells were incubated in 5% CO₂ incubator at 37 °C and 95% humidity with medium change every second day. Upon reaching 85% confluency, the cells were washed with cold phosphate-buffered saline (PBS) and detached with 5 mL TrypLE™ Express Enzyme (Fisher Scientific). Then TrypLE™ was neutralized with ice-cold culture medium containing 10% FBS and the cells pelleted by centrifuging at 200 × g (Eppendorf AG, Hamburg, Germany) for 5 min. The cells were re-suspended and counted, processed for the seeding of 3D MCF-7 culture to obtain MCF-7 cell spheroids. The cell suspension containing 4 × 10⁴ and 3 × 10⁵ cells/mL in 200 µL was seeded into a 96-well plate for determination of cell response to rHuEPO using the MTT and neutral red retention time (NRRT) assays, respectively. Cell suspensions containing 1.25 × 10⁵ cells/mL were seeded into a 6-well plate for the determination of cell response to rHuEPO using TBE, caspase activity, DNA fragmentation, and AO/PI staining assays and cell cycle analysis.

The MCF-7 cell spheroid from 3D culture used in the study were generated using the ultra-low adhesion technique (ULAT) technique. 300 µL cell suspension containing 1.6 × 10⁴ cells/mL were dispensed in each well of a 96-well conical-bottomed microplate (Nunc® MicroWell™ 96-well polystyrene plates, USA). The cells were cultured in RPMI1640 medium (Gibco^TM cell culture media, Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (iFBS) and 1% of penicillin/streptomycin (Gibco^TM, Fisher Scientific, USA), and incubated in the 5% CO₂ incubator at 37 °C and under 95% humidity. After treatment with rHuEPO, single cells obtained from the disruption of MCF-7 cell spheroids were manipulated similarly to monolayer cells from 2D cultures. The cells were then subjected to the determination of cell response to rHuEPO treatment via cell viability assays; MTT, NRRT, TB, DNA fragmentation, and AO/PI staining caspases and cell cyle analysia.

SARS‐CoV‐2 S1, S2, and N recombinant proteins were purchased from RayBiotech (Peachtree Corners, GA). RvD1, RvD2, and RvD1 EIA were purchased from Cayman Chemicals. RvD1 and RvD2 were stored and prepared before each experiment as previously published.⁶ Gibco cell culture media, fetal bovine serum (FBS), and supplements were purchased from Thermo Fisher Scientific. IL‐8 ELISA kits were purchased from PeproTech (London).