Lung epithelial cell line H292 (ATCC) was cultured in RPMI (Thermo Fisher Scientific) with 10% v/v FBS, 10 mM HEPES and sodium pyruvate (Thermo Fisher Scientific), nonessential amino acids (Thermo Fisher Scientific) and additional 20 mM L-glutamine (Thermo Fisher Scientific) without antibiotics. For seeding, medium was aspirated and replaced with 1 ml ice cold PBS (Sigma-Aldrich or Thermo Fisher Scientific) twice. Cells were lifted using 0.25% Trypsin-EDTA (Thermo Fisher Scientific) solution that was neutralized with complete media. Cells were centrifuged (200 g, 5 min), resuspended in the complete media and counted. All stimulations, pre-treatments are described in the main text and figure legends. 16–20 hr prior stimulation medium was replaced to serum-free RPMI.
Hepes and sodium pyruvate
Manufactured by Thermo Fisher Scientific
HEPES and sodium pyruvate are two commonly used reagents in cell culture and laboratory applications. HEPES is a buffer compound that helps maintain a stable pH in cell culture media, while sodium pyruvate is an energy source for cells. Both of these products are essential for supporting the growth and maintenance of cell lines in a laboratory setting.
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Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Plvx ires puro, by Takara Bio (1 mentions)
Snap tag, by New England Biolabs (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Non essential amino acid (neaa), by Thermo Fisher Scientific (1 mentions)
Anti vimentin, by Novus Biologicals (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Saponin, by Merck Group (1 mentions)
4 protocols using hepes and sodium pyruvate
1
Culturing Lung Epithelial H292 Cells
2
Lung Explant Culture Protocol
3
Immortalized Mouse Embryonic Fibroblasts
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The vim+/+ and vim−/− mEFs were derived from vim+/+ and vimentin-null mice and immortalized by stable expression of SV40 large T antigen (kindly provided by J. Eriksson, Abo Akademi University, Turku, Finland). Cells were cultured in DMEM with 25 mM Hepes and sodium pyruvate (Life Technologies) supplemented with 10% FBS, 1% penicillin/streptomycin, and nonessential amino acids. All cell cultures were maintained at 37°C and 5% CO2.For the reconstitution of vimentin protein expression in vim−/− mEFs, the vimentin cDNA was expressed from pLVX-IRES-neo (Clonetech) by lentiviral transduction. To express the SNAP-vimentin fusion protein, the vimentin cDNA was subcloned into pLVX-IRES-puro (Clontech) with the addition of a SNAP tag (NEB). After selection of the transduced cells with the appropriate antibiotic for 1 wk vimentin expression was assessed by Western blotting using mouse anti-vimentin antibody (Sigma, clone V9, V6630) as described before (Gan et al., 2016 (link)).
4
Immortalized Mouse Embryonic Fibroblasts Protocol
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Mouse embryonic fibroblasts were derived from wild-type and vimentin null mice and immortalized by stable expression of SV40 large T-antigen (kindly provided by J. Ericsson, Abo Akademi University, Turku, Finland)25 (link). Cells were grown in 1X DMEM (Life Technologies; Catalog no: MT10013CV) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, Catalog no: SH3008803), 1% penicillin-streptomycin (Gibco) and nonessential amino acid (Life Technologies), and 10 mM HEPES and sodium pyruvate (Life Technologies) at 37 °C with 5% CO2. Cells were plated at a density of 10,000 cells/gel or less.
For immunofluorescence experiments, cells were fixed with 4% paraformaldehyde (Affymetrix) followed by 5% BSA and 1% Saponin (Sigma) for blocking and permeabilization. Primary antibodies were Alexa- Fluor 647 phalloidin (Invitrogen Catalog no: A22287), anti-vimentin (Novus Biologicals Catalog no: NB300-223), and dapi (Sigma Catalog no: D9542). Alpha-tubulin rat antibody (Bio-Rad- Catalog no: MCA77G) at a concentration of 1:200 was used for detecting microtubules.
For immunofluorescence experiments, cells were fixed with 4% paraformaldehyde (Affymetrix) followed by 5% BSA and 1% Saponin (Sigma) for blocking and permeabilization. Primary antibodies were Alexa- Fluor 647 phalloidin (Invitrogen Catalog no: A22287), anti-vimentin (Novus Biologicals Catalog no: NB300-223), and dapi (Sigma Catalog no: D9542). Alpha-tubulin rat antibody (Bio-Rad- Catalog no: MCA77G) at a concentration of 1:200 was used for detecting microtubules.
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