Bronchial smooth muscle cells
(BSMC, Lonza CC-2576), small airway epithelial cells (SAEC, Lonza
CC-2547), or HepG2 cells (ATCC HB-8065) were seeded in 96-well plates
at a density of 25,000 per well. Culture of these cells throughout
the protocol utilized incubation at 37 °C, 5% CO2 in
a humidified atmosphere, with BSMC and SAEC cells cultured in cell-specific
media (Lonza) and HepG2 cells cultured in RPMI 1640 medium containing
Glutamax (Gibco) and 10% fetal bovine serum. Plates containing BSMC
cells were collagen coated to assist with cell adhesion. Following
an overnight settling period, media was replaced with fresh media,
with, or without, compound. Compound concentrations tested were 1,
3, 10, 30, and 100 μM, in duplicate. The cells were incubated
with compound for 24 h. Control (1% DMSO) and positive standards terfenadine-
and salmeterol-treated groups were included for both cell lines. Following
the 24 h compound incubation period, 10 μL of MTS reagent (Promega
G5421) was added to 50 μL cell media and adhered cells, and
the plates were incubated for 45 min at 37 °C, 5% CO2 in a humified atmosphere. The reaction was stopped with 12.5 μL
of 10% SDS, plates briefly shaken, and absorbance (490 nm) read using
a Spectramax M5e plate reader (Molecular Devices). Percentages of
viable cells were calculated as: (test sample Abs 490 nM-mean blank
Abs 490 nm)/(mean control Abs 490 nM-mean blank Abs 490 nm) ×
100.
(BSMC, Lonza CC-2576), small airway epithelial cells (SAEC, Lonza
CC-2547), or HepG2 cells (ATCC HB-8065) were seeded in 96-well plates
at a density of 25,000 per well. Culture of these cells throughout
the protocol utilized incubation at 37 °C, 5% CO2 in
a humidified atmosphere, with BSMC and SAEC cells cultured in cell-specific
media (Lonza) and HepG2 cells cultured in RPMI 1640 medium containing
Glutamax (Gibco) and 10% fetal bovine serum. Plates containing BSMC
cells were collagen coated to assist with cell adhesion. Following
an overnight settling period, media was replaced with fresh media,
with, or without, compound. Compound concentrations tested were 1,
3, 10, 30, and 100 μM, in duplicate. The cells were incubated
with compound for 24 h. Control (1% DMSO) and positive standards terfenadine-
and salmeterol-treated groups were included for both cell lines. Following
the 24 h compound incubation period, 10 μL of MTS reagent (Promega
G5421) was added to 50 μL cell media and adhered cells, and
the plates were incubated for 45 min at 37 °C, 5% CO2 in a humified atmosphere. The reaction was stopped with 12.5 μL
of 10% SDS, plates briefly shaken, and absorbance (490 nm) read using
a Spectramax M5e plate reader (Molecular Devices). Percentages of
viable cells were calculated as: (test sample Abs 490 nM-mean blank
Abs 490 nm)/(mean control Abs 490 nM-mean blank Abs 490 nm) ×
100.
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