hCMEC/D3 cells were cultured on Lab-Tek™ Permanox™ 8-well chamber slides coated with calf skin collagen (Sigma-Aldrich, UK), prior to immunostaining according to standard protocols [79 (link), 81 (link)] and using primary antibodies directed against Nrf2 (1:500, Novus Biologicals Ltd., Abingdon, UK), occludin (1:200, Thermo-Fisher Scientific, UK), claudin-5 (1:200, Thermo-Fisher Scientific, UK) and zona occludens-1 (ZO-1; 1:100, Thermo-Fisher Scientific, UK). Nuclei were counterstained with DAPI (Sigma-Aldrich, UK). Images were captured using an LSM880 confocal laser scanning microscope (Carl Zeiss Ltd., Cambridge, UK) fitted with 405, 488 and 561 nm lasers and a × 63 oil immersion objective lens (NA, 1.4 mm, working distance, 0.17 mm). Images were captured with ZEN imaging software (Carl Zeiss Ltd., UK) and analysed using ImageJ 1.51 h (National Institutes of Health, USA).
Calf skin collagen
Manufactured by Merck Group
Sourced in United Kingdom, Austria
Calf skin collagen is a raw material derived from the hides of calves. It is a natural protein that can be used in various laboratory applications. The core function of calf skin collagen is to provide a source of collagen for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (2 mentions)
Millicell ers 2 voltohmmeter, by Merck Group (2 mentions)
Pet transwell inserts, by Greiner (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Humanht 12 v4.0 expression beadchip, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Clariostar spectrophotometer, by BMG Labtech (1 mentions)
Lvis microplate, by BMG Labtech (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Bz x700 automated microscope, by Keyence (1 mentions)
Quasar 570, by Biosearch Technologies (1 mentions)
6 protocols using calf skin collagen
1
Immunostaining of hCMEC/D3 Cells
2
Collagen Quantification via Sirius Red
3
Transcriptomic Analysis of hCMEC/D3 Cells
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hCMEC/D3 cells were grown on 6-well plates coated with calf-skin collagen (Sigma–Aldrich, Gillingham, UK) and collected in TRIzol (Thermo-Fisher Scientific, UK) as described previously [2 (link)]. Total RNA was extracted using a TRIzol Plus RNA purification kit (Thermo-Fisher Scientific, UK) and quantified using a CLARIOstar spectrophotometer equipped with an LVis microplate (BMG Labtech GmbH, Germany).
Hybridization experiments were performed by Macrogen Inc. (Seoul, Republic of Korea) using Illumina HumanHT-12 v4.0 Expression BeadChips (Illumina Inc., San Diego, CA) as described previously [2 (link)].
Hybridization experiments were performed by Macrogen Inc. (Seoul, Republic of Korea) using Illumina HumanHT-12 v4.0 Expression BeadChips (Illumina Inc., San Diego, CA) as described previously [2 (link)].
4
Evaluating Barrier Function of hCMEC/D3 Cells
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Paracellular permeability and transendothelial electrical resistance (TEER) were measured on 100% confluent hCMEC/D3 cultures polarised by growth on 24-well plate polyethylene terephthalate (PET) transwell inserts (surface area: 0.33 cm2, pore size: 0.4 μm; Greiner Bio-One GmbH, Kremsmünster, Austria) coated with calf-skin collagen and fibronectin (Sigma–Aldrich, UK). The permeability of hCMEC/D3 cell monolayers to 70-kDa FITC-dextran (2 mg/ml) was measured as described previously [31 (link)–33 (link)]. TEER measurements were performed using a Millicell ERS-2 Voltohmmeter (Millipore, Watford, UK) and were expressed as Ω cm2. In all cases, values obtained from cell-free inserts similarly coated with collagen and fibronectin were subtracted from the total values. In some cases, barrier integrity was tested by challenge with bacterial lipopolysaccharide (LPS). Confluent hCMEC/D3 monolayers were treated with TMAO or TMA for 12 h, whereupon LPS (Escherichia coli O111:B4; 50 ng/ml, comparable to circulating levels of LPS in human endotoxemia [34 ]) was added for a further 12 h, without wash out. Barrier function characteristics were then interrogated as described above.
5
Quantifying SARS-CoV-2 Infection in Huh7 Cells
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In a 12-well plate, calf skin collagen (Sigma-Aldrich, St. Louis, MO) coated glass cover slips (18 mm round #1, Neuvitro, ThermoFisher Scientific) were seeded with 2E+05 Huh7 cells/well 24 h prior to infection. Cells were infected with HCoV-OC43 (MOI = 0.025) diluted in “infection medium” containing either 0.25, 0.1, 0.025 or 0.01 μM RDV or DMSO vehicle control for 1 h at 32 °C. After 1 h, monolayers were washed and infection medium containing the previously described DMSO or RDV was added. After 6 days at 32 °C, coverslips were fixed with 3.7% formaldehyde in PBS for 10 min, washed with PBS and stored in 70% ethanol until staining. 48 unique oligonucleotide probes against ORF1a coupled with Quasar 570 were fabricated by LGC Biosearch Technologies. Fixed coverslips were FISH stained and nuclei were counterstained with Hoechst 33258 (ThermoFisher Scientific) according to the protocol provided by LGC. Coverslips were imaged on a Keyence BZX-700 automated microscope. Images were acquired with the same settings and cell quantitation was performed using the Keyence Hybrid Cell Count analysis package (Matsuda et al., 2018 (link)).
6
Evaluating Endothelial Barrier Integrity
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Paracellular permeability and transendothelial electrical resistance (TEER) were measured on 100 % confluent hCMEC/D3 cultures polarised by growth on 24-well plate polyethylene terephthalate (PET) transwell inserts (surface area: 0.33 cm 2 , pore size: 0.4 μm; Greiner Bio-One GmbH, Kremsmünster, Austria) coated with calf-skin collagen and fibronectin (Sigma-Aldrich, UK). The permeability of hCMEC/D3 cell monolayers to 70 kDa FITC-dextran (2 mg/ml) was measured as described previously [31] [32] [33] . TEER measurements were performed using a Millicell ERS-2 Voltohmmeter (Millipore, Watford, UK) and were expressed as Ω.cm 2 . In all cases, values obtained from cell-free inserts similarly coated with collagen and fibronectin were subtracted from the total values. In some cases, barrier integrity was tested by challenge with bacterial lipopolysaccharide (LPS). Confluent hCMEC/D3 monolayers were treated with TMAO or TMA for 12 h, whereupon LPS (Escherichia coli O111:B4; 50 ng/ml, comparable to circulating levels of LPS in human endotoxemia [34] ) was added for a further 12 h, without wash-out. Barrier function characteristics were then interrogated as described above.
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