Primary mouse lung microvascular endothelial cells (MLECs) and complete mouse endothelium cell medium were both purchased from Cell Biologics, (Chicago, IL). Cells were maintained in 10-cm plastic dishes pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Chicago, IL). For some experiments, MLECs were cultured with serum from lean or obese mice (10 μl serum/ml complete mouse endothelium cell medium containing 1% FBS). At pre-specified time points, cell lysates were collected for later analysis. All in vitro studies were performed using cells from passage 3–4.
Gelatin based coating solution
Manufactured by Cell Biologics
Sourced in United States
Gelatin-based coating solution is a liquid product designed to create a uniform, biocompatible coating on various lab equipment and surfaces. It is formulated to enhance cell attachment and proliferation in in vitro cell culture applications.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Ccl 149, by American Type Culture Collection (2 mentions)
Rat primary tracheal epithelial cells, by Cell Biologics (2 mentions)
Complete epithelial cell medium, by Cell Biologics (2 mentions)
F 12k medium, by American Type Culture Collection (2 mentions)
Vero e6 cell, by American Type Culture Collection (2 mentions)
Complete human epithelial cell media, by Cell Biologics (2 mentions)
Epithelial cell medium, by Cell Biologics (2 mentions)
Primary small iecs, by PELOBiotech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mlecs, by Cell Biologics (1 mentions)
Egm 2 mv microvascular endothelial cell growth medium 2 bulletkit, by Lonza (1 mentions)
C57bl 6 mouse primary brain microvascular endothelial cells, by Cell Biologics (1 mentions)
Complete mouse endothelial cell medium w kit, by Cell Biologics (1 mentions)
Dasatinib, by Merck Group (1 mentions)
Insulin transferrin selenium, by Thermo Fisher Scientific (1 mentions)
Q4951, by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
Cc 5036, by Lonza (1 mentions)
Endothelial cell medium (ecm), by Cell Biologics (1 mentions)
19 protocols using gelatin based coating solution
1
Primary Mouse Lung Endothelial Cells
2
Mammalian Cell Culture Conditions
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Vero E6 cells (CRL-1586, American Type Culture Collection [ATCC]) and 293FT/hACE2+TMPRSS (17 (link)) were cultured in Dulbecco’s Modified Eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Rat lung epithelial cells L2 (CCL-149, ATCC) were cultured in F-12K Medium (ATCC) supplemented with 10% FBS at 37°C with 5% CO2. Rat primary tracheal epithelial cells (Cell Biologics) were grown on culture flasks or plates pre-coated with gelatin-based coating solution (Cell Biologics) in Complete Epithelial Cell Medium (Cell Biologics) at 37°C with 5% CO2.
3
Culturing Primary Colonic Cells
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Human primary colonic epithelial cells and human primary colonic microvascular endothelial cells were cultured following manufacturer’s instructions (Cell Biologics, Chicago, IL, USA). Cells were thawed and cultured in a T75 flask coated with gelatin-based coating solution (Cell Biologics) with either complete human epithelial cell media (Cell Biologics) or EGM-2 MV microvascular endothelial cell growth medium-2 BulletKit (Lonza, Walkersville, MD). Media was changed after initial plating, every 48 h when cells were <70% confluent and every 24 h when cells were >70% confluent. Cells were harvested at confluency for experimentation.
4
Culturing Primary Mouse Dermal Lymphatic Endothelial Cells
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Mouse primary dermal lymphatic endothelial cells (LECs) from the C57BL/6 mouse strain were purchased from Cell Biologics (#C57-6064L, Chicago, USA) and cultured in tissue culture flasks precoated with gelatin-based coating solution (#6950, Cell Biologics) in Culture Complete Mouse Endothelial Cell Medium (#M1168, Cell Biologics). Cells were passaged after reaching a confluence of approx. 80%.
5
Culturing Mouse Brain Endothelial Cells
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C57BL/6 mouse primary brain microvascular endothelial cells were obtained directly from Cell Biologics (Catalogue No. C57-6023, Chicago, IL). Cells were grown in 75 cm2 flasks coated with a gelatin-based coating solution (Cell Biologics, Chicago, IL). Endothelial cell media containing vascular endothelial growth factor, endothelial cell growth supplement, heparin, epidermal growth factor, hydrocortisone, L-glutamine, an antibiotic-antimycotic solution, and fetal bovine serum (Complete Mouse Endothelial Cell Medium w/ Kit, Cell Biologics, Chicago, IL) was used to grow the endothelial cells to approximately 80% confluency.
6
Investigating PDIA3 and TGF-β1 in Kidney Fibroblasts
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Human primary kidney fibroblasts (Cell biologics) were plated at 3 × 106 cells per well of a 6-well plastic culture dish plate precoated with gelatin-based coating solution (Cell biologics). Cells were maintained in fibroblast medium with added FCS (Cell biologics). Cells were grown at 37°C in 5% CO2. At 24 hours before being treated with the protein of interest, medium was swapped for serum-free medium to ensure a “serum-starved” phenotype to minimize baseline activation of cells. Cells were then treated with PDIA3 (10 ng/mL) ± TGF-β1 (10 ng/mL). Cells were collected for qPCR 72 hours later.
7
Isolation and Senescence Depletion of Type II Alveolar Epithelial Cells
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AECs were isolated by magnetic associated cell sorting (MACS) by negative selection of CD45+ (Miltenyi, cat#130-052-301, dilution 10 µL per 10 million cells) and CD31+ (Miltenyi, cat#130-097-41, dilution 10 µL per 10 million cells) cells and followed by positive selection of CD326+ cells (Miltenyi, cat# 130-118-075, clone caa7-9G8, dilution 5 µL per 10 million cells). This method typically yields approximately 85% purity of prosurfactant-C+ cells as measured by flow cytometry. Isolated type II AECs were cultured with DMEM/F12 medium containing 10% FBS, 1.25 g BSA, 100 U/mL penicillin/streptomycin, and 1x Insulin-Transferrin-Selenium (Gibco, 41400045). Type II AECs were seeded in tissue culture plates coated with gelatin-based coating solution (Cell Biologics, 6950).
To deplete senescent cells, type II AECs were cultured with 20 nM dasatinib (Sigma, SML2589) and 5 µM quercetin (Sigma, Q4951) or 200 nM dasatinib and 50 µM quercetin for 48 h.
To deplete senescent cells, type II AECs were cultured with 20 nM dasatinib (Sigma, SML2589) and 5 µM quercetin (Sigma, Q4951) or 200 nM dasatinib and 50 µM quercetin for 48 h.
8
Characterizing HUVEC Viability on Gelatin Patches
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Human umbilical vein endothelial
cells (HUVECs) were cultured in endothelial cell growth basal medium-plus
(Lonza, no. CC-5036) according to the manufacturer’s specification.
Patches were cut to a diameter of 6 mm using a biopsy punch and coated
with gelatin-based coating solution (Cell Biologics, #6950) via incubation
at room temperature for 10 min. HUVEC cells (5 × 103) were seeded onto patches in 20 μL droplets and incubated
for 1 h to aid attachment before patches were covered with media and
incubated for 24 h at 37 °C. Following incubation, viability
was assessed via immunofluorescent staining with 2 μM calceinAM
and 8 μM ethidium homodimer. Z-stack images
were acquired at 10× magnification using the A1 Nikon Confocal
Imaging System (Nikon Corporation, Tokyo, Japan). Z-stacks were composed of ≥25 images at 15 μm intervals.
cells (HUVECs) were cultured in endothelial cell growth basal medium-plus
(Lonza, no. CC-5036) according to the manufacturer’s specification.
Patches were cut to a diameter of 6 mm using a biopsy punch and coated
with gelatin-based coating solution (Cell Biologics, #6950) via incubation
at room temperature for 10 min. HUVEC cells (5 × 103) were seeded onto patches in 20 μL droplets and incubated
for 1 h to aid attachment before patches were covered with media and
incubated for 24 h at 37 °C. Following incubation, viability
was assessed via immunofluorescent staining with 2 μM calceinAM
and 8 μM ethidium homodimer. Z-stack images
were acquired at 10× magnification using the A1 Nikon Confocal
Imaging System (Nikon Corporation, Tokyo, Japan). Z-stacks were composed of ≥25 images at 15 μm intervals.
9
Culturing Primary Small Intestinal Epithelial Cells
10
Mouse Brain Microvascular Endothelial Cell Culture
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Mouse primary brain microvascular endothelial cells (MBEC) were purchased from Cell Biologics, IL, United States (Catalog# C57-6023; Lot# 070613T2MP) and cultured according to the manual, but with no heparin (as it interferes with EV uptake Atai et al., 2013 (link); Christianson et al., 2013 (link)). Briefly, T25 flasks were pre-coated with Gelatin-based coating solution (Cell Biologics), 106 cells seeded in the Endothelial Cell Medium (Cell Biologics) and passaged 1:2 upon confluence. Low passages (1–4) have been used in this study.
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