Cath.-a-differentiated (CAD) cells were purchased from Sigma-Aldrich and cultured in DMEM/F12 medium (Gibco) supplemented with 8% fetal calf serum, 1% L-Glutamine, and 1% penicillin-streptomycin. Two to four hours prior to imaging, CAD cells were plated on coverslips coated overnight at 4 °C with 10 µg/mL laminin (Sigma-Aldrich). DMEM/F12 medium without phenol red (Gibco) supplemented with 15mM HEPES was used for live-cell imaging. Cell lines were also routinely tested for mycoplasma using the Universal Detection Kit (ATCC). PFN1 KO cells were generated with CRISPR/Cas9 as previously described (28 (link)). CAD cells were transfected with plasmid DNA via electroporation as previously described (28 (link)) or with LipoD293 (SignaGen, “Hard-To-Transfect Mammalian Cell” protocol). Cells expressing HaloTag constructs were incubated overnight with 10–100 nM Janelia Fluor 646 HaloTag ligand (Promega) (61 (link)). EGFP-NM2A knock-in MEFs were generated from mice (62 (link)), and isolated and cultured as previously described (40 (link)).
Universal detection kit
Manufactured by American Type Culture Collection
The Universal Detection Kit is a laboratory product designed to detect the presence of a wide range of target organisms in samples. It provides a standardized and efficient method for identifying the presence or absence of specific targets, without interpretation or extrapolation of the results.
Automatically generated - may contain errors
Lab products found in correlation
Laminin, by Merck Group (2 mentions)
Dmem f12 medium without phenol red, by Thermo Fisher Scientific (2 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
293t cell, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Vero e6, by American Type Culture Collection (2 mentions)
Lipod293, by SignaGen (1 mentions)
Janeliafluor 646 halotag ligand, by Promega (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Usa wa1 2020, by BEI Resources (1 mentions)
4 protocols using universal detection kit
1
Culturing and Labeling Neuronal Cell Lines
2
CRISPR Knockout of Profilin-1 in CAD Neuroblastoma Cells
3
Establishment of hACE2-Expressing Cell Line
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Vero E6 (ATCC, CRL-1586) and 293T cells (ATCC, CRL-3216; kind gift of Dr. Viviana Simon), were maintained in DMEM (Corning) supplemented with 10% FB (Peak Serum) and penicillin/streptomycin (Corning) at 37°C and 5% CO2. hACE2-293T cells were generated for this study. Briefly, 293T cells were transduced with a lentiviral vector expressing human ACE2. Puromycin resistant cells with hACE2 surface expression were sorted after staining with AlexaFluor 647-conjugated goat anti-hACE2 antibodies. Cells were then single-cell-cloned and screened for their ability to support SARS-CoV-2 replication. All cell lines used in this study were regularly screened for mycoplasma contamination using the Universal Detection Kit (ATCC, 30-1012K).
4
SARS-CoV-2 Infection in ACE2-Expressing Cells
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Vero E6 (ATCC, CRL-1586) and 293T cells (ATCC, CRL-3216; kind gift of Dr. Viviana Simon), were maintained in DMEM (Corning) supplemented with 10% FB (Peak Serum) and penicillin/streptomycin (Corning) at 37°C and 5% CO2. hACE2-293T cells were generated for this study. Briefly, 293T cells were transduced with a lentiviral vector expressing human ACE2. Puromycin resistant cells with hACE2 surface expression were sorted after staining with AlexaFluor 647-conjugated goat anti-hACE2 antibodies. Cells were then single-cell-cloned and screened for their ability to support SARS-CoV-2 replication. All cell lines used in this study were regularly screened for mycoplasma contamination using the Universal Detection Kit (ATCC, 30-1012K). Cells were infected with SARS-CoV-2, isolate USA-WA1/2020 (BEI Resources NR-52281) under biosafety level 3 (BSL3) containment in accordance to the biosafety protocols developed by the Icahn School of Medicine at Mount Sinai. Viral stocks were grown in Vero E6 cells as previously described (Amanat et al., 2020 (link)), and were validated by genome sequencing.
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