One step mycoplasma detection kit

Vero, HEK293T and HeLa cells were all obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (Biological Industries, Kibbutz, Israel) supplemented with a final concentration of 10% fetal bovine serum (FBS) (Biological Industries) and 1% penicillin-streptomycin-glutamine (Gibco, Carlsbad, USA). All cells were treated with MycAway^TM elimination reagent (Yeason, Shanghai, China), followed by testing using One-Step Mycoplasma Detection Kit (Yeason) to make sure they were Mycoplasma free. Both the T. gondii RHΔku80 strain and EGFP-RHΔku80 strain were serially passaged as tachyzoites in Vero cells in our own laboratory as previously described [13 (link)]. Briefly, tachyzoites were collected in the supernatant of centrifugation of spontaneously ruptured T. gondii-infected Vero cells at 300g for 5 min at room temperature. They were further cleaned by passing through a syringe filter of 5-μm pore size (Millipore, Darmstadt, Germany). Parasites were used to infect fresh Vero cells at a multiplicity of infection (MOI) of 10:1 after quantification using a hemocytometer. Six-week-old BALB/c mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China), and housed in a ventilated cage kept under a conditioned temperature of 25 °C with a light/dark cycle of 14 h/10 h.

Parental T. gondii RHΔku80Δhxgprt (here referred as RHΔku80) and subsequent strains were cultivated in confluent monolayers of human foreskin fibroblasts (HFF) in Dulbecco’s modified Eagle medium (DMEM) (Biological Industries, Israel) supplemented with 2.5% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin-glutamine (Gibco). All strains and host cell lines were tested negative for Mycoplasma sp. with the One-Step Mycoplasma Detection Kit (Yeason, China). The cells were cultured in a humidified incubator with 5% CO₂ at 37°C. Tachyzoites used in experiments were freshly purified with a syringe filter of 5-μm pore size (Millipore, Germany).

The HNSCC cell lines 6-10B and Tu 686 were purchased from FuHeng Cell Center (Shanghai, China). The cell lines mentioned above were cultured in complete medium: RPMI Medium 1640 (Gibco, USA) supplemented with 10% fetal calf serum (Gibco, USA), penicillin (100 units/ml) and streptomycin (100 μg/ml) at 37°C in a thermostatic incubator with 5% CO₂. All cells were validated using DNA short tandem repeat analysis each six months and tested for mycoplasma contamination using One-Step Mycoplasma Detection Kit (Yeason, Shanghai, China) each four weeks.
Functional assays were conducted in two groups: a negative group (sh-NC) and an shRNA knock-down group (sh-PER3). Cell proliferation was detected by the Cell Counting Kit-8 (CCK-8) and clone formation assays. In brief, cells were inoculated into 96-well plates (1000 cells/well). At each time point (1st, 2nd, 3rd, 4th, and 5th day), 10 μl of CCK-8 solution (Yeason, Shanghai, China) was added to the sextuplicate wells. The plates were incubated for 1.5 h, and the optical density values were detected. For clone formation assays, cells were seeded in a six-well plate (1000 cells/well) with the culture medium refreshed every 3 days for 2 weeks. Following the 2 weeks, the cells were washed with PBS, fixed, stained, and counted.