In order to stain live cells intracellularly while maintaining plasma membrane integrity, we adapted a cationic lipid-aided intracellular staining protocol described by Weill et al.68 (link). A mix of either primary anti-filaggrin (G-20; Santa Cruz Biotechnology, Dallas, TX, USA) or isotype control (goat; BD, Franklin Lakes, NJ, USA) antibody and secondary antibody (anti-goat Alexa 488; Life Technologies/ThermoFisher Scientific, Waltham, MA, USA) was prepared in low calcium ([Ca2+] = 0.06 mM) keratinocyte medium (KBM-2; Lonza) at 1:200 dilution. Lipofectamine 2000 cationic lipid reagent (Life Technologies/ThermoFisher Scientific, Waltham, MA, USA) was added at 0.5 µl/ml and incubated for 15 min. The cell culture medium was replaced by Lipofectamine 2000 containing a mix for 2–4 h and then washed off multiple times. The cell nuclei were stained using Hoechst (NucBlue Live Ready Probes, Life Technologies/ThermoFisher Scientific, Waltham, MA, USA). Calcium-supplemented KBM-2 medium (Lonza, Basel, Switzerland) ([Ca2+] = 1.5 mM or 5 mM) was added, and imaging (one Z-stack every hour; distance between each axial layer with a Z-stack: 300 nm) was carried out on the Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) over a period of 24–48 h following the first calcium switch, with temperature and humidity control.
Kbm 2
Manufactured by Lonza
Sourced in Switzerland, United States
The KBM-2 is a laboratory equipment designed for cell culture applications. It serves as a basic incubator, providing a controlled environment for the growth and maintenance of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Dksfm, by Thermo Fisher Scientific (2 mentions)
Opsys mr microplate reader, by Thermo Fisher Scientific (1 mentions)
Plx4032, by Cayman Chemical (1 mentions)
Rspo1, by Thermo Fisher Scientific (1 mentions)
Rspo2, by Thermo Fisher Scientific (1 mentions)
Vivaspin protein concentrator, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Nheks, by Lonza (1 mentions)
Anti flg goat g20, by Santa Cruz Biotechnology (1 mentions)
Hoechst nucblue, by Thermo Fisher Scientific (1 mentions)
Ni no3 2, by Merck Group (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Anti rabbit alexa 568, by Thermo Fisher Scientific (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
L ascorbic acid 2 phosphate sesquimagnesium salt hydrate, by Merck Group (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
12 protocols using kbm 2
1
Intracellular Staining of Live Cells
2
AM Epithelial Cell Proliferation Assay
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AM epithelial cells were seeded into 96-well culture plates in a density of 1 × 104 cells/well in 100 µl of defined KGM-2 medium with five independent replicates per treatment condition. After 24 h, the cells were washed once with PBS and starved in Keratinocyte basal Medium 2 (KBM2, Lonza) overnight. Then Rspo1 (PeproTech) and Rspo2 (PeproTech) were administrated to the starved AM epithelial cells at concentrations of 0, 5, 10, and 20 ng/ml, respectively. After 72 h, 10 µl of CCK-8 reagent (Cell Counting Kit-8 assay, BioLegend) was added into each well and incubated at 37 °C for 2 h. For drug resistance testing, AM epithelial cells were seeded into 96-well plates (5 × 104 cells/well) and treated with PLX4032 (Cayman Chemical Company) for 48 h and the cell viability was determined by Cell Count Kit-8. The absorbance at 450 nm wavelength was detected using an OPSYS Mr microplate reader (Thermo Fisher).
3
In vitro antimicrobial assay
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hTCEpi cells were trypsinated, washed, and lysed with sterile saline by three cycles of freeze and thaw. Preparation of <10-kD cytosolic lysate fractions and antimicrobial assays against P. aeruginosa (cytotoxic strain 6206) were performed as described previously (Tam et al., 2012 (link)). In brief, lysates (1 mg/ml) normalized by BCA assays were fractionated at 4°C using a series of water-prerinsed Vivaspin protein concentrators with membrane cutoff at 100, 30, or 10 kD (GE Healthcare). All filtrates, including the <10-kD fractions, were normalized to the initial volume with saline. The inoculum was consisted of a P. aeruginosa culture at 108 colony-forming units (CFUs)/ml (OD650 reading at 0.1) in keratinocyte basal medium 2 (KBM-2; Lonza) diluted 100-fold in <10-kD lysate fractions. The mixtures were incubated for 3 h at 37°C followed by 10-fold serial dilutions by PBS and plated onto tryptic soy agar plates. After incubation overnight, the number of colonies in the agar plates was counted and adjusted for the dilution factor to derive viable bacterial counts (CFU/ml).
4
Isolation and Culture of Primary Airway Mesenchymal Cells
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The primary AM or OKC cells were isolated as previously described [23 (link)]. The suspended primary AM cell were seeded in the 0.1% gelatin-coated (PRIMARY CELL SOLUTION, PCS-999-027) culture dishes (1×106) in defined Keratinocyte Basal Medium-2 (KBM-2, LONZA, CC-3103) supplemented with KGMⓇ-2 SingleQuotsⓇ (LONZA, CC-4152) at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO2). After 48 h, the non-adherent cells were removed, and fresh KGMⓇ-2 media were replenished every 3 days. Early passages of primary cells were cryopreserved, and less than six passages were used for further experiments. For calcium imaging experiment or histological analysis, AM cells were seeded in the KGMⓇ-2 media with DMSO (0.1% v/v), Bay-k8644 (10 nM) or VPM (10 µM) and cultured for 12 h.
5
Isolation and Treatment of Primary Human Corneal Epithelial Cells
6
Primary Human Corneal Epithelial Cell Responses to Bacterial Challenges
7
Culturing and Maintaining Human Eye Cell Lines
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Human retinal pigment epithelial cells (ARPE-19 cell line, ATCC CRL-2302) were cultured using DMEM/F12 media (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, ThermoFisher Scientific, Waltham, MA, USA) [8 (link),36 (link)]. Vero cells (ATCC CCL-81) were cultured in DMEM medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, while the human corneal epithelial cells (HUCL cell line) were maintained in a defined keratinocyte-serum-free medium (KBM-2, Lonza, Basel, Switzerland) in a humidified 5% CO2 incubator at 37 °C [9 (link),37 (link)]. PVP-I reagent (ReadyPrep PVP-I 10% solution, Medline, Northfield, IL, USA) was used for the study.
8
Nickel-Induced Keratinocyte Differentiation
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Normal human epidermal keratinocytes (NHEKs) (purchased from Lonza) were cultured in monolayers in a dedicated medium (Lonza, KBM-2) at the Ca2+ level of 0.06 mM. To stimulate differentiation and FLG expression, a calcium switch was conducted over a period of 24 h by replacing the culture media with fresh media adjusted to a 1.5 mM final calcium concentration. A Ni(NO3)2 (Sigma) solution was added to achieve various final concentrations (10 μM, 100 μM and 1 mM). Doses were chosen based on MTT test results (Supplementary Figure S1 ). After 24 h of incubation, the cells were fixed, permeabilized and immunostained with anti-FLG antibodies (Anti-FLG goat G20 (Santa Cruz), and secondary anti-goat Alexa-488 and anti-rabbit Alexa-568 (Life Technologies) antibodies were used. Staining was carried out in PBS and nuclei were visualized by Hoechst (NucBlue, Life Technologies). The slides were coversliped with Mowiol 4-88 (Sigma). Data acquisition was carried out on the Zeiss 780 inverted confocal microscope. Images from three separate experiments were analysed; KHG diameter and integrated intensity from the signal were measured using Fiji: ImageJ program (Abramoff, 2007 (link)). For the statistical analysis the Mann–Whitney U test was used.
9
Characterization of Oral Cancer Cell Lines
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As a translational science cell line project, this study did not require IRB review at our institution. CA‐9‐22 are derived from squamous cell carcinoma of the human tongue and grown in RPMI supplemented with 10% FBS at 37°C in 5% CO2 (ThermoFisher, Waltham, MA). MSK‐Leuk1 cells were isolated from a dysplastic leukoplakic lesion adjacent to a squamous cell carcinoma of the tongue. These were a kind gift from Dr. Peter Sacks and are grown in Keratinocyte Growth Medium (KBM‐2, Lonza, Walkersville, MD) supplemented with bovine pituitary extract, epidermal growth factor, insulin, and hydrocortisone at 37°C in 5% CO2. Rhek‐1A cells are SV40‐immortalized human epidermal keratinocytes grown in DMEM supplemented with 10% FBS at 37°C in 5% CO2 (ThermoFisher). Cell line authentication was carried out by STR genotyping by the Fragment Analysis Facility at Johns Hopkins University followed by BLAST searches of the ATCC, CLIMA, and DSMZ databases. MSK‐Leuk1 cells and Rhek‐1A cells were phenotyped and found not to match any existing cell line records in any database. CA‐9‐22 cells were phenotyped and found to match existing cell line records for CA‐9‐22 cells from the Riken Cell Bank in Japan.
10
Immortalized Human Corneal Epithelial Cells
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Telomerase immortalized human CE cells (hTCEpi; CE/keratinocytes) from Danielle M Robertson (James Jester's laboratory) and primary human keratocytes were a kind gift from Dr. Shukti Chakravarty, Departments of Ophthalmology and Pathology, NYU School of Medicine. The CE cells were seeded and maintained in keratinocyte basal medium‐2 (KBM‐2) (#CC3103, Lonza BioScience) or containing KGM‐2 Bullets (Singlequote kit; #CC4152; Lonza BioScience) as keratinocyte complete growth media (KGM). Primary corneal keratocytes cells were maintained in Complete Media of DMEM/F12 (#11039021, Gibco) supplemented with 5% FBS, 1% Penicillin–Streptomycin solution (#30‐001‐CI, Corning) and 1 mM L‐ascorbic acid 2‐phosphate sesquimagnesium salt hydrate (#A8960, Sigma).
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