Mm hepes

Manufactured by Thermo Fisher Scientific

MM HEPES is a laboratory buffer solution used to maintain a stable pH environment in cell culture and biochemical applications. It is formulated using the HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer compound.

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DMEM 25 mM Hepes GlutaMAX-I 25 mM HEPES HEPES

6 protocols using mm hepes

Culturing and Cryopreserving Colorectal Cancer Cell Lines

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Three human colorectal cancer cell lines
(COLO 205, HCT 116, and SW620) and one normal human fetal small intestine
cell line (HIEC6) were obtained from the American Type Culture Collection
(ATCC). COLO205, HCT116, and SW620 were grown in RPMI-1640 (Gibco)
supplemented with 10% fetal bovine serum (FBS), while HIEC-6 was grown
in OptiMEM 1 reduced serum medium (Gibco) supplemented with 20 mM
HEPES (Gibco), 10 mM GlutaMAX (Gibco), 10 ng/mL epidermal growth factor
(EGF) (Gibco), and FBS to a final concentration of 4%. All cells were
maintained at 37 °C with 5% CO₂.
Cells were
rinsed with warm phosphate-buffered saline (PBS) before being trypsinized
with TrypLE Express Enzyme (1×) (Gibco) for 5–15 min at
37 °C with 5% CO₂. The harvested material was then
spun at 1000 rpm for 5 min, rinsed once with warm PBS, and then resuspended
in ice-cold PBS. After cell count, replicates of 2 × 10⁸ cells were pelleted and frozen at −80 °C until further
use.

Wu Z., Bonneil É., Belford M., Boeser C., Ruiz Cuevas M.V., Lemieux S., Dunyach J.J, & Thibault P. (2022). Proteogenomics and Differential Ion Mobility Enable the Exploration of the Mutational Landscape in Colon Cancer Cells. Analytical Chemistry, 94(35), 12086-12094.

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Cell Culture and Treatment with Targeted Therapies

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LIM1215, HCA7 and HEK-293T cell lines were cultured in DMEM/F-12 + L-Glutamine + 15 mM HEPES (Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 1% penicillin/streptomycin (Gibco) at 37°C with 5% CO₂. LIM1215 cells were obtained from the Ludwig Institute for Cancer Research, New York, NY, USA and HCA7 and HEK-293T cells were obtained from the American Type Culture Collection (ATCC).
Cetuximab (Erbitux^®) was purchased from Merck Serono, lapatinib (Tykerb/Tyverb^®) from GlaxoSmithKline, and trastuzumab (Herceptin^®) and pertuzumab (Perjeta^®) from Roche. Gefitinib (ZD1839), GDC-0941, trametinib (GSK1120212) and afatinib (BIBW2992) were purchased from Selleck Chemicals. NRG1 was purchased from Peprotech.

Bosch-Vilaró A., Jacobs B., Pomella V., Asbagh L.A., Kirkland R., Michel J., Singh S., Liu X., Kim P., Weitsman G., Barber P.R., Vojnovic B., Ng T, & Tejpar S. (2016). Feedback activation of HER3 attenuates response to EGFR inhibitors in colon cancer cells. Oncotarget, 8(3), 4277-4288.

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Thioglycollate-Elicited Macrophage Protocol

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Bone marrow transplantation was performed, as described. Mice were put on a HCD for 10 weeks and subsequently injected with thioglycollate medium (3%, Fischer). Four days after injection, mice were sacrificed and the peritoneum was flushed twice with 10 ml PBS to collect peritoneal macrophages. Flushed thioglycollate-elicited macrophages were cultured in RPMI-1640 25 mM HEPES, 2 mM l-glutamine, 10% FCS, penicillin (100 U/ml), and streptomycin (100 μg/ml) (all Gibco) and allowed to adhere for 3 h. Oil Red O staining (0.3% in 60% isopropanol, Sigma) was performed to determine lipid accumulation. For gene expression experiments, macrophages adhered overnight.

Hoeksema M.A., Gijbels M.J., Van den Bossche J., van der Velden S., Sijm A., Neele A.E., Seijkens T., Stöger J.L., Meiler S., Boshuizen M.C., Dallinga-Thie G.M., Levels J.H., Boon L., Mullican S.E., Spann N.J., Cleutjens J.P., Glass C.K., Lazar M.A., de Vries C.J., Biessen E.A., Daemen M.J., Lutgens E, & de Winther M.P. (2014). Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions. EMBO Molecular Medicine, 6(9), 1124-1132.

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Culturing and Spiking H209 SCLC Cells

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The human SCLC cell line, H209 (ATCC^® HTB-172) was obtained from the American Type Culture Collection (Manassas, VA, USA) and used as positive control in IF experiments. H209 cells were cultured in RPMI 1640 (Gibco-BRL Life Technologies, Grand Island, NY), supplemented with 10% fetal bovine serum (Gibco-BRL), 2 mM L-glutamine (Gibco-BRL), 10 mMHepes (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1.5 g/L NaHCO₃ (Sigma-Aldrich), 4.5 g/L glucose (Sigma-Aldrich) and 50 mg/ml penicillin/streptomycin (Gibco-BRL). Cells were maintained in a humidified atmosphere of 5% CO₂, at 37 °C. To determine the sensitivity of the method, H209 cells were spiked in peripheral blood obtained from healthy individuals, and the PBMCs and the corresponding, obtained after Ficoll-Hypaque density centrifugation, and cytospins were prepared as above. All experiments were performed during the logarithmic growth phase of the cell line.

Messaritakis I., Stoltidis D., Kotsakis A., Dermitzaki E.K., Koinis F., Lagoudaki E., Koutsopoulos A., Politaki E., Apostolaki S., Souglakos J, & Georgoulias V. (2017). TTF-1- and/or CD56-positive Circulating Tumor Cells in patients with small cell lung cancer (SCLC). Scientific Reports, 7, 45351.

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Albumin-Free Murine HSC Expansion

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HSCs were cultured using a PVA-based albumin-free culture system as
described in a previous report (Wilkinson
et al., 2019). Briefly, HSCs were cultured in media composed of
F12 medium (Gibco), 10 mg/L insulin (Gibco), 6.7 μg/L sodium selenite
(Sigma), 2 mg/L ethanolamine (Sigma), 1% P/S and L-Glutamine (Gibco), 10 mM
HEPES (Gibco), 0.1% poly-vinyl alcohol (PVA, Sigma), 100 ng/ml murine TPO
(PeproTech), 10 ng/ml murine SCF (PeproTech), and human holo-transferrin
(Sigma) with defined concentrations ranging from 5 μg/L to 500 mg/L.
Fifty sorted Lin⁻ Sca-1⁺ c-Kit⁺CD48⁻ CD150⁺ CD34⁻ HSCs
were used as input, and cultured in human fibronectin coated 96-well
microplates (R&D systems) for 18–21 days. Complete medium change
was initiated 5 days after culture started, and performed every 2–3
days. Cultured cells were split 1:3 into new plates at ~90% confluency, and
harvested for analyses and transplantation when they reach confluency for
the second time.

Zhang D., Gao X., Li H., Borger D.K., Wei Q., Yang E., Xu C., Pinho S, & Frenette P.S. (2022). The microbiota regulates hematopoietic stem cell fate decisions by controlling iron availability in bone marrow. Cell stem cell, 29(2), 232-247.e7.

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Proteomic analysis of HAP1 cells

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All of the chemicals were of the highest purity commercially available and used without further purification. Ammonium bicarbonate, calcium chloride, phosphate buffered saline (PBS), Tris-HCl, Triton X-100, sucrose, mannitol, ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), formic acid (FA), dithiothreitol (DTT), iodoacetamide (IAA), and fetal bovine serum (FBS) were obtained from Sigma–Aldrich (Milan, Italy). IMDM (Iscove’s Modified Dulbecco’s Medium containing L-glutamine and 25 mM HEPES) was obtained from Gibco-Thermo Fisher Scientific. Human HAP1 cells were from Horizon Discovery (Cambridge, United Kingdom UK). Modified porcine trypsin and chymotrypsin were purchased from Promega (Milan, Italy). Water and acetonitrile (OPTIMA® LC/MS grade) for LC/MS analyses were provided from Fisher Scientific (Milan, Italy).

Pittalà M.G., Saletti R., Reina S., Cunsolo V., De Pinto V, & Foti S. (2020). A High Resolution Mass Spectrometry Study Reveals the Potential of Disulfide Formation in Human Mitochondrial Voltage-Dependent Anion Selective Channel Isoforms (hVDACs). International Journal of Molecular Sciences, 21(4), 1468.

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