Kaluza software gallios

GC-2spd cells subjected to hypoxic treatment for 48 h or treated with CoCl₂ for 24 h were stained using the PE Annexin V Apoptosis Detection Kit (BD Pharmingen; BD Biosciences, San Jose, CA, United States) according to the manufacturer’s protocol and analyzed using a flow cytometer and Kaluza software (Gallios; Beckman Coulter, Inc. Boulevard Brea, CA, United States).

The cDNAs of titin, ryanodine receptor, and Kv1.4 were subcloned into mammalian expression vectors containing GFP (ATUM, Newark, CA). Human embryonic kidney 293F cells (Thermo Fisher Scientific, Tokyo) were utilized in transfection with these vectors. The 293F and transfected cells were cultured in Dulbecco’s Modified Eagle Medium with 4.5 g/L D-glucose, L-glutamine supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. The stable transfected cells were taken from the culture flask with 0.05% ethylenediaminetetraacetic acid/phosphate-buffered saline.
To test for the presence of antibodies, 5.0 × 10⁶ culture cells were incubated on ice for 1 hour with the patients’ serum (1:100). Then the cells were washed and incubated with PE-labeled secondary anti-human IgG (1:1,000) antibodies (Thermo Fisher Scientific, Tokyo) for 30 minutes. After incubating with 7-aminoactinomycin D (Thermo Fisher Scientific, Tokyo), the cells were applied to a flow cytometer and analyzed with Kaluza software (Gallios, Beckman Coulter, Brea, CA).