Collagen Coating for Tissue Culture

Abstract

This protocol is intended for collagen coating of tissue culture dishes and Transwell® multiple well plates. The attached methods document is a formal version of the information included here.Disclaimer: The information presented here has been reviewed and approved for publication by the US Environ do not necessarily represent Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.

Characterizing the extracellular vesicle proteomic landscape of the human airway using in vitro organotypic multi-cellular models

“…All cells were passaged onto tissue culture dishes (Techno Plastic Products, #93150 and 93100) coated with bovine type I collagen solution (Advanced BioMatrix, San Diego, CA, #5005). 69 All cell types were authenticated by short tandem repeat (STR) profiling using the ATCC Human Cell Authentication Service (ATCC #135-XV; results included in supplemental information ) and tested negative for Mycoplasma spp. contamination (Universal Mycoplasma Detection Kit, ATCC #30-1012K).…”

“…Detailed cell culture methods for the H441 cell line is available through the online, publicly available Nature Portfolio, Protocol Exchange. 69 , 70 , 71 , 72 In brief, IMR90 were maintained in Advanced-RPMI growth medium (A-RPMI; ThermoFisher #12633020) supplemented with 5% FBS (ThermoFisher, #16000044, certified), 0.5% Penicillin/Streptomycin (P/S; ThermoFisher, #15140122) and 4 mM GlutaMAX (ThermoFisher #35050061)). Fibroblasts: IMR90 cells (human, female, lung fibroblasts; ATCC #CCL-182, Batch #64155514) represented the fibroblasts and were obtained from ATCC.…”

“…Detailed cell culture methods for the IMR90 cell line is available through the online, publicly available Nature Portfolio, Protocol Exchange. 69 , 70 , 71 , 72 In brief, IMR90 were maintained in Advanced-RPMI growth medium (A-RPMI; ThermoFisher #12633020) supplemented with 5% FBS (ThermoFisher, #16000044, certified), 0.5% Penicillin/Streptomycin (P/S; ThermoFisher, #15140122) and 4 mM GlutaMAX (ThermoFisher #35050061)). Lung microvascular endothelial cells: HULEC-5a cells (hereafter referred to as “HULEC”; human, male, lung microvascular endothelial cells, ATCC #CRL-3244, Batch #70025430) represented the endothelial cells and were obtained from ATCC.…”

“…Detailed cell culture methods for the HULEC-5a cell line is available through the online, publicly available Nature Portfolio, Protocol Exchange. 69 , 70 , 71 , 72 HULEC were maintained in complete HULEC growth medium (MCDB-131 (Gibco #10372019) supplemented with 10% FBS, 1% P/S, 10 mM GlutaMAX, 10 ng/mL human Epidermal Growth Factor (Fisher Scientific, #PHG0311), and 1μg/mL hydrocortisone (MilliporeSigma, #H0888)).…”

Culture of HULEC-5a Cells

“…A face shield should be worn while handling liquid nitrogen and cell vials that have been recently removed from liquid nitrogen storage as they may explode unexpectedly. HULEC cells should be thawed and sub-cultured on collagen coated tissue culture plastics (McNabb et al, 2019). HULEC cells used within an APD range of 3-12 have high levels of reproducibility within subcultured experiments.…”