Inferior lacrimal glands were removed aseptically and finely minced. Cell isolation was carried out as previously described [36 (link)]. Briefly, the tissue was enzymatically digested by collagenase (350 U/ml; Worthington Biochemical Corp, Lakewood, NJ), DNase I (40 U/ml; Roche) and hyaluronidase (300 U/ml; Sigma-Aldrich) for 45 min at 37 °C, and the digests were filtered through a 100 μm cell strainer and then centrifuged at 200g for 5 min. Cells were resuspended in HepatoStim culture medium supplemented with epidermal growth factor (EGF; 5 ng/mL; Corning, Tewksbury, MA). The LGACs were seeded on Matrigel® (growth factor reduced, Corning) coated wells at a density of 5 × 105 cells/cm2, and incubated at 37 °C with 5% CO2.
Hepatostim culture medium
Manufactured by Corning
HepatoStim culture medium is a specialized cell culture medium designed to support the growth and maintenance of hepatocytes, which are the primary functional cells of the liver. The medium is formulated to provide the necessary nutrients and growth factors to promote the survival and differentiation of hepatocytes in vitro.
Automatically generated - may contain errors
2 protocols using hepatostim culture medium
1
Isolation and Culture of Lacrimal Gland Acinar Cells
2
Amylase Activity Evaluation of SGSC Differentiation
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During the differentiation of SGSCs
into SG epithelial cells, changes in the function of SGSCs were evaluated
by amylase activity. The SGSCs were seeded on the substrates at 3
× 104 cells/well, and then cultured until the appropriate
spheroid formation was reached. Fresh medium was added every 3 days.
The SGSCs in 2D monolayers and 3D spheroids were stimulated with epinephrine
(Sigma, Epi, 10 μM) or isoproterenol (Sigma, ISO, 1 μM)
in Hepato-STIM culture medium (Corning) for 45 min before measurement.
The amylase activity was measured from media collected at 5 days using
an α-amylase activity assay kit (Abcam) with 2-chloro-p-nitrophenol
linked to maltotriose as the chromogenic substrate, in accordance
with the manufacturer’s instructions. The α-amylase activity
of individual samples was directly proportional to increases in absorbance
at 405 nm using a standard laboratory plate reader. Absorbance (OD)
was measured in triplicate from three independent experiments, and
data were normalized to the total number of counted cells. The negative
control was culture medium without cells.
into SG epithelial cells, changes in the function of SGSCs were evaluated
by amylase activity. The SGSCs were seeded on the substrates at 3
× 104 cells/well, and then cultured until the appropriate
spheroid formation was reached. Fresh medium was added every 3 days.
The SGSCs in 2D monolayers and 3D spheroids were stimulated with epinephrine
(Sigma, Epi, 10 μM) or isoproterenol (Sigma, ISO, 1 μM)
in Hepato-STIM culture medium (Corning) for 45 min before measurement.
The amylase activity was measured from media collected at 5 days using
an α-amylase activity assay kit (Abcam) with 2-chloro-p-nitrophenol
linked to maltotriose as the chromogenic substrate, in accordance
with the manufacturer’s instructions. The α-amylase activity
of individual samples was directly proportional to increases in absorbance
at 405 nm using a standard laboratory plate reader. Absorbance (OD)
was measured in triplicate from three independent experiments, and
data were normalized to the total number of counted cells. The negative
control was culture medium without cells.
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