Mycoplasma detection kit

HEK293T cells were cultured in DMEM (GIBCO 11995065) containing phenol red, 4 mM L-glutamine, 110 mg L⁻¹ sodium pyruvate, 4.5 g L⁻¹ D-glucose, and supplemented with 10% FBS (GIBCO 26140079) and 2% Pen/Strep (Thermo Fisher 15070063). Cells lines were tested for mycoplasma contamination before use via the Mycoplasma Detection Kit (SouthernBiotech 13100-01). Cells were maintained in a humidified atmosphere of 5% CO₂ and 37°C and were passaged every 2-3 days into 10 cm polystyrene coated plates (VWR 10062-880) upon reaching high density.

All cell lines were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 in a humidified incubator, and were routinely checked for mycoplasma with a Mycoplasma Detection Kit (SouthernBiotech, Cat# 13100–01). To generate cells stably expressing wild-type Cas9 endonuclease (HEK293T-Cas9), HEK293T cells (ATCC) were transduced with lentiviral particles produced from lentiCas9-Blast (Addgene, #52962) and selected for blasticidin resistance.

To test BSL-4 cell supernatants or viral stocks for mycoplasma contamination, we used the QuickExtract™ DNA Extraction Solution (Epicentre Biotechnologies, Madison, WI, USA) in combination with heat inactivation to extract mycoplasma DNA for PCR analysis from the samples. QuickExtract™ DNA Extraction Solution is used to extract genomic DNA from diverse samples for PCR amplification. Cell lysis and DNA extraction include a 6-min incubation at 65 °C followed by a 2-min incubation at 98 °C. We adapted this protocol to our needs by replacing the second heat step with a 10-min incubation at 120 °C in the Advanced Dry Block Heater (VWR), which we use for heat inactivation. Briefly, 50 µL of cell supernatant or virus stock solution were mixed with 200 µL QuickExtract™ DNA Extraction Solution in a sealed screw top tube and incubated for 6 min at 65 °C, followed by 10 min at 120 °C. The Mycoplasma Detection kit from SouthernBiotech (Birmingham, AL, USA) was used according to the manufacturer’s instructions. M. orale DNA is included in the kit as a positive control and leads to a 503 bp PCR fragment. Mycoplasma-infected samples will generate PCR products between 448 and 611 bp, depending on the mycoplasma species that is present. An internal negative control (270 bp) is included to prevent false negatives due to PCR inhibitors.

Immortalized human bronchial epithelial cells from a ΔF508 homozygous cystic fibrosis patient (CFBE41o- [36 (link)]) were cultured at 37°C and 5% CO₂ in minimal essential medium (MEM) (Gibco) supplemented with 2 mM l-glutamine, 5 U/ml penicillin, and 5 μg/ml streptomycin (Sigma); 0.5 μg/ml plasmocin prophylactic (InvivoGen); and 10% fetal bovine serum (FBS; Gemini Bio-Products). CFBE41o- cells were seeded at confluence on Transwell permeable MEMbrane supports (Costar) and differentiated at the air-liquid interface for 1 week prior to use or were seeded at confluence and polarized on 40-mm-diameter glass coverslips (Fisher Scientific) for 1 week prior to use. CFBE41o- cells are not on the list of commonly misidentified cells, and cells were tested quarterly for the presence of mycoplasma using a mycoplasma detection kit (Southern Biotech).
Primary human bronchial epithelial cells were obtained from explanted lungs of adult bronchiectasis patients with informed consent under a protocol approved by the Institutional Review Board at the University of Pittsburgh (REN16100171). All relevant ethical regulations were complied with. Epithelial cells were seeded on permeable MEMbrane supports and differentiated at the air-liquid interface prior to use.

Mycoplasma test (13100–01, Mycoplasma Detection Kit, SouthernBiotech) was performed on the 4 cell lines used for the in vitro experiments. All cell lines were mycoplasma negative.