African green monkey kidney Vero E6 cells (C1008) were plated at 2 × 104 cells/well density in opaque (white) tissue culture 96‐well plates (Greiner Bio‐One) at the same time as the addition of the drug treatments and virus treatments and cultured in MEM (Gibco) supplemented with 2% HI‐FBS. Human liver epithelial THLE‐2 cells (CRL2706, ATCC) were plated at 3 × 103 cells/well density in 96‐well plates coated with bronchial epithelial cell growth (BEBM) complete medium with fibronectin (0.01 mg/ml; Biological Industries), bovine collagen Type I (0.03 mg/ml; Stem Cell Technologies) and bovine serum albumin (0.01 mg/ml; Sigma‐Aldrich). The BEBM complete medium consisted of BEGM Bullet Kit (Lonza) excluding gentamicin/amphotericin and epinephrine but additionally supplemented with EGF (5 ng/ml), phosphoethanolamine (70 ng/ml), and 10% FBS (Biowest). Human cardiomyocyte AC16 cells (SCC‐09, Millipore) were plated at 2 × 103 cells/well density in 96‐well uncoated plates and cultured in complete AC16 medium—DMEM/F12 (Life Technologies) supplemented with l ‐glutamine (2 mm; Life Technologies), 12.5% FBS (Biowest), and 1% penicillin–streptomycin (Life Technologies). All cell cultures were incubated in a humidified atmosphere, at 37°C with 5% CO2 atmosphere.
Bovine collagen type 1
Manufactured by STEMCELL
Bovine collagen Type I is a purified protein derived from bovine sources. It provides a structural framework for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Sartorius (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Biowest (1 mentions)
Begm bullet kit, by Lonza (1 mentions)
Human fibronectin, by Thermo Fisher Scientific (1 mentions)
Anti stat1, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Beyozol, by Beyotime (1 mentions)
Anti stat2, by Abcam (1 mentions)
Beyofast sybr green qpcr mix, by Beyotime (1 mentions)
Anti jak1, by Proteintech (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Penicillin streptomycin, by Beyotime (1 mentions)
4 protocols using bovine collagen type 1
1
Cell Culture Optimization for Compound Screening
2
Molecular Assays for Antiviral Signaling
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LB broth (catalog# ST156, Shanghai, China), BeyoECL Plus (catalog# P0018S), Bradford protein assay kit (catalog# P0006), BeyoFast™ SYBR Green qPCR Mix (2X, catalog# D7260), penicillin–streptomycin (catalog# C0222), fetal bovine serum (FBS, catalog# C0225), Beyozol (catalog# R0011), BeyoRT™ II cDNA synthesis reagent (catalog# D7168M), RIPA lysis buffer (catalog# P0013C), and bovine serum albumin (BSA, catalog# ST025-5 g) were purchased from Beyotime Biotechnology. Phosphate-buffered saline (PBS) was purchased from Thermo Fisher Scientific (pH 7.4, catalog# 10010072). Dulbecco's modified Eagle’s medium (DMEM) was purchased from Thermo Fisher Scientific (catalog# 11995073). BEGM media with SingleQuot kit additives were purchased from Lonza (catalog# CC-3170, Walkersville, MD). Human fibronectin (catalog# PHE0023) was purchased from Thermo Fisher Scientific (catalog# PHE0023, Waltham, MA). Bovine collagen type I was purchased from STEMCELL (catalog# 07001, STEMCELL). Anti-IFN-α polyclonal antibody (catalog# 66162-1-Ig, Proteintech), Anti-IFN-β polyclonal antibody (catalog# 27506-1-AP, Proteintech), Anti-JAK1 (rabbit monoclonal, catalog# ab133666), anti-STAT1 (rabbit monoclonal, catalog# ab234400), anti-GAPDH (rabbit monoclonal, catalog# ab8245), and anti-STAT2 (rabbit monoclonal, catalog# ab32367) were purchased from Abcam.
3
Preparation of Differentiated Macrophages from THP-1 Cells
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Prior to all experiments, live/dead cell count was determined by 4% trypan blue
dye exclusion for viability. Cell populations with a minimum of 95% viability
were used for the experiments. HBEC line (BEAS-2B, ATCC CRL-9609) was seeded
(1 × 106) onto type I bovine collagen (StemCell Technologies,
Vancouver, BC)-coated T-75 flasks. Cells were maintained in serum-free LHC-9
medium (Gibco) containing penicillin/streptomycin (100 U/ml; Gibco) and
amphotericin B (2 µg/ml; Sigma) in a humidified chamber at 37˚C/5%
CO2 until 80% confluent. Immortalized human monocytic cell line
(THP-1, ATCC TIB-202) was maintained in suspension culture in Roswell Park
Memorial Institute medium (RPMI-1640, Gibco) containing 10% of heat inactivated
FBS (Atlanta Biologics) and supplemented with HEPES buffer (10 mM; Gibco),
sodium pyruvate (1 mM; Sigma), D-Glc (4.5 g/l; Sigma), sodium bicarbonate
(1.5 g/l; Sigma), penicillin/streptomycin (100 U/ml; Gibco) and amphotericin B
(2 µg/ml; Sigma). Cells were maintained in a humidified chamber at 37˚C/5%
CO2. 2-3 × 106 cells were stimulated with 10 nM
phorbol 12-myristate 13-acetate (PMA, Sigma) diluted in RPMI-1640 with 1% FBS
for 24 h to differentiate monocytes into macrophages and incubated for an
additional 24 h.
dye exclusion for viability. Cell populations with a minimum of 95% viability
were used for the experiments. HBEC line (BEAS-2B, ATCC CRL-9609) was seeded
(1 × 106) onto type I bovine collagen (StemCell Technologies,
Vancouver, BC)-coated T-75 flasks. Cells were maintained in serum-free LHC-9
medium (Gibco) containing penicillin/streptomycin (100 U/ml; Gibco) and
amphotericin B (2 µg/ml; Sigma) in a humidified chamber at 37˚C/5%
CO2 until 80% confluent. Immortalized human monocytic cell line
(THP-1, ATCC TIB-202) was maintained in suspension culture in Roswell Park
Memorial Institute medium (RPMI-1640, Gibco) containing 10% of heat inactivated
FBS (Atlanta Biologics) and supplemented with HEPES buffer (10 mM; Gibco),
sodium pyruvate (1 mM; Sigma), D-Glc (4.5 g/l; Sigma), sodium bicarbonate
(1.5 g/l; Sigma), penicillin/streptomycin (100 U/ml; Gibco) and amphotericin B
(2 µg/ml; Sigma). Cells were maintained in a humidified chamber at 37˚C/5%
CO2. 2-3 × 106 cells were stimulated with 10 nM
phorbol 12-myristate 13-acetate (PMA, Sigma) diluted in RPMI-1640 with 1% FBS
for 24 h to differentiate monocytes into macrophages and incubated for an
additional 24 h.
4
Glycogen Accumulation Staining in Cells
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For fibroblasts or HepG2 cells staining, round coverslips Ø12mm (0.13-016mm) (ThermoScientific-Menzel) were previously coated with 0.1% gelatin, and for iHeps staining coverslips were previously coated with collagen solution 1:45 (Type I bovine collagen, Stem Cell Cat#04902). Samples were fixed with 4% paraformaldehyde (PFA) solution (Paraformaldehyde 16%; AlfaAesar, Cat#43368) in PBS for 15 minutes at room temperature. Fixed cells samples were staining for the identification of glycogen accumulation using Periodic Acid-Schiff Kit (Sigma, Cat#395B), according to manufacturer's indications. Briefly, fixed cells were rinsed with water for 1 minute and incubated in Periodic Acid for 5 minutes at room temperature. After several changes of distilled water to rinse the slides, they were incubated with Schiff’s reagent for 15 minutes at room temperature. Finally, samples were washed with water and visualized under light microscope (Nikon ECLIPSE Ts2R-FL). Images were taken with a Leica DFC70000T camera.
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