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We will submit a grant proposal before May 1st. Please help us with methodology.
Growing/Passaging Calu-3 Cells (*Below is not describing Air-Interfaced Culture (AIC))
Calu-3 cells will be grown in 35 x 10 mm cell culture Petri dishes. RPMI Medium 1640 with 10% Fetal Bovine Serum (FBS) will be used as a growth medium for the cells. The cells will be passaged to new 35 x 10 mm dishes upon reaching >90% confluency. Passing will be performed through the use of 0.25% Trypsin (1X) EDTA to dissociate the cells, while a small portion of the trypsinized cell solution will be transferred to the new dish.
The Calu-3 cells will be incubated in Forma Series II Water Jacketed CO2 Incubator. The incubator will have conditions set to 95% O2, 5% CO2, and 37 degrees C.
Transfecting Calu-3 Cells (*Not for AIC)
24 hours before transfection, the Calu-3 cells will be plated at 125,000 cells per well in a 24-well cell culture plate. Liposome mediated transfection will then be utilized to transfect the desired DNA into the Calu-3 cells. The Invitrogen Lipofectamine 3000 transfection kit will be used to perform the transfection. The transfected cells will incubate for 2-4 days following transfection. Cells with <50 passages will be ideal for transfecting.
Article Comparing Liquid Covered Culture (LCC) of Calu-3 Cells to Air-Interfaced Culture (AIC). This paper found that AIC produced Calu-3 cells with morphology that more closely resembled airway epithelium.
Video Outlining how to establish LCC culture of Calu-3 Cells With Transwell System, and response of this LCC to a Virus: https://www.jove.com/video/50157/establishing-liquid-covered-culture-polarized-human-airway-epithelial
How to produce SARS-CoV-2 Spike Protein, hACE2 and Decoy Receptor: https://www.ncbi.nlm.nih.gov/pubmed/29240734
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