Mycoseq mycoplasma detection assay

To generate the HEK293T cell line ectopically expressing 2x-FLAG-Casp11 mutant, the plasmid encoding this protein was packaged into lentivirus by transfecting the vector (2 μg) along with psPAX2 (2 μg), and pMD2.G (1 μg) using Fugene HD transfection reagent (Promega) into HEK293T cells. After 2 d, the supernatants were filtered using a 0.45 μm filter, then used to infect HEK293T cells. After 48 hr, the cells expressing the indicated constructs were selected with puromycin (1 μg/mL; Sigma-Aldrich). Cell lines were tested to be Mycoplasma-free by use of the standard MycoSEQ Mycoplasma detection assay (ThermoFisher Scientific).

HEK293T cells were purchased from ATCC, grown in complete DMEM (supplemented with 10% v/v FBS, 10 mM HEPES, 10 mM sodium pyruvate, 1% Penicillin/Streptomycin), and maintained in a 37°C incubator with 5% CO₂. Murine bone marrow progenitors were harvested from femurs and hip bones of 8–12-week-old C57BL/6 or Casp11^-/- mice and differentiated into BMDMs using 30% L929-conditioned complete DMEM for 7 d as previously described (Bjanes et al., 2021 (link)). Cell lines were tested to be Mycoplasma-free by use of the standard MycoSEQ Mycoplasma detection assay (Thermo Fisher Scientific).