The 125-mL or 1-L Celstir spinner flasks (catalog nos. 356876 and 356884) were assembled and siliconized using Sigmacote (Millipore Sigma, catalog no. SL2-100ML) according to the manufacturers’ instructions. Cytodex 1, 2,200 cm2/L (Cytiva, catalog no. 17044801), Cytodex 3 (Cytiva, catalog no. 17048502), collagen-coated (Corning, catalog no. 3786), and BioNOC II (Chemglass Life Sciences, catalog no. CLS-1344-01) were rehydrated and sterilized in siliconized Erlenmeyer flasks according to their respective manufacturer’s instructions. After this microcarrier comparison, 6 g/L of Cytodex 1 was consistently used. Microcarriers were rinsed with DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific, catalog no. 15140122) before being transferred to a sterilized bioreactor using a siliconized 5-mL glass pipette. The working volume was increased to 70% using the medium above and allowed to equilibrate at 37°C in 5% CO2 and a 60 rpm stirring rate for a minimum of 3 h. Unless stated otherwise, bioreactors were inoculated with 5 cells per microcarrier. After 24 h, the working volume was increased to 100%.
Collagen coated
Manufactured by Corning
Sourced in United States
Corning's Collagen-coated lab equipment is a specialized product designed for cell culture applications. The collagen coating provides a natural extracellular matrix-like environment to support cell attachment, proliferation, and differentiation. This product is intended to facilitate in vitro cell studies and research.
Automatically generated - may contain errors
3 protocols using collagen coated
1
Microcarrier Comparison for Cell Culture
2
Base Editor Protein Transfection in NIH/3T3 Cells
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NIH/3T3 cells were seeded on 48-well collagen-coated BioCoat plates (Corning) in an antibiotic-free DMEM medium and transfected at ∼75% confluency. Base editor proteins were incubated with 1.1-fold molar excess of the indicated sgRNA at 25 °C for 5 min. The complex was then incubated with 1.4 μl Lipofectamine 3000 (Thermo Fisher) and transfected according to the manufacturer's protocol for plasmid delivery. P3000 reagent was not used because its addition leads to protein precipitation and a reduction in base-editing efficiency. Unless otherwise noted, BE protein was added to a final concentration of 400 nM (based on a total well volume of 275 μl).
3
Caco-2 Cell Barrier Integrity Assay
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Caco-2 cells were seeded at a density of 104 cells/well into a collagen-coated (50 µg/mL; Corning, Corning, NY, USA) upper chamber of a 6-well transwell filter (pore diameter, 0.4 µm; Costar). The transepithelial electric resistance (TEER) was measured with a voltmeter (Millicels; Millipore, Bedford, MA, USA) before the stimulation (T = 0) and daily during 60 h after stimulation with LPS (10 µg/mL; #L4005, Sigma-Aldrich), PG (60 µg/mL) and the combination of the two stimuli (LPS + PG). The TEER value was calculated by subtracting the blank value. Finally, the percentage of change in TEER was evaluated with time 0 for each condition.
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