Hepes buffer

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HEPES buffer is a chemical buffer solution used in cell culture and other biological applications. It is designed to maintain a stable pH within a physiological range, typically between 7.2 and 7.6, to support the growth and maintenance of cells in vitro.

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HEPES (4332 citations) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (21 citations) HEPES buffer (1 M) (16 citations) Hepes 1 M buffer solution (6 citations) HBSS–HEPES buffer (3 citations) HEPES-Hank’s Balanced Salt Solution (HBSS) buffer (2 citations) HEPES buffer pH7.2-7.5 (2 citations) 1M Hepes buffer solution (2 citations) Hepes buffer (pH 7.2) (2 citations) HEPES 0.9% NaCl buffer (2 citations)

569 protocols using hepes buffer

Intracellular Nitric Oxide Measurement

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Intracellular NO was measured in real time using the NO-specific fluorescence probe DAF-FM DA solution (Sigma-Aldrich). DAF-FM DA diffuses freely across the membrane and is hydrolyzed by intracellular esterases, resulting in the formation of DAF-FM. Intracellular DAF-FM reacts with the NO oxidation product N₂O₂, which generates the stable highly fluorescent derivative DAF-FM triazole. Cells were washed with modified HEPES buffer (20 mM HEPES buffer [Gibco] with 5 mM glucose, 50 µM l-arginine, and 0.1% BSA, pH 7.0–7.4), incubated with 5 µM DAF-FM DA in modified HEPES buffer for 30 min at room temperature, washed again, and finally incubated in modified HEPES buffer for 30 min at 37°C in the absence or presence of 1 mM L-NMMA. Fluorescence (emission wavelength, 485 nm; excitation wavelength, 538 nm) was measured at 37°C from 1 to 10 min using a fluorescence microtiter plate reader (Synergy HTX Multi-Mode Reader, BioTek). eNOS activity was expressed as the VEGFA-dependent increase in fluorescence per microgram of cellular protein. To determine the cellular protein content, the same cells were lysed in 1% (vol/vol) Triton X-100 and analyzed for protein content with the BCA protein detection kit. DAF-FM DA experiments were repeated three times. Within each experiment, four wells were used for each NO measurement.

Ninchoji T., Love D.T., Smith R.O., Hedlund M., Vestweber D., Sessa W.C, & Claesson-Welsh L. (2021). eNOS-induced vascular barrier disruption in retinopathy by c-Src activation and tyrosine phosphorylation of VE-cadherin. eLife, 10, e64944.

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Breast Cancer Cell Line Culture Conditions

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MCF-10A and MCF-7 cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 media containing 2.5 mM L-glutamine and 15 mM HEPES buffer (Life Technologies, Grand Island, NY, USA). MDA-MB-231 and T47D cells were cultured with RPMI-1640 medium containing 2.5 mM L-glutamine and 25 mM HEPES buffer (Life Technologies). All cells were cultured at 37°C with 5% CO₂. For standard culture conditions, medias were supplemented with 10% fetal bovine serum (FBS) (heat inactivated, HyClone, Logan, UT, USA) penicillin-streptomycin-glutamine, 5.0 μg/mL of insulin-transferrin-selenium-X (ITS-X) (Life Technologies), and 2.5 nM epidermal growth factor (EGF), recombinant human (BD Biosciences, San Jose, CA, USA). In the case of treatment with C-6 or its analogs, low serum medias were used (2% fetal bovine serum).

Vaden R.M., Gligorich K.M., Jana R., Sigman M.S, & Welm B.E. (2014). The small molecule C-6 is selectively cytotoxic against breast cancer cells and its biological action is characterized by mitochondrial defects and endoplasmic reticulum stress. Breast Cancer Research : BCR, 16, 472.

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Platelet Activation and Concentration

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Expired platelet packs were donated from the University of Wisconsin Blood Bank, aliquoted, and stored at 4°C for processing on the day of receipt. Platelet aliquots were centrifuged at 2000 × g for 12 minutes, and the supernatant was collected and saved (referred to as “Plasma,” P) or was used to re-suspend un-activated platelets (referred to as “Platelets+Plasma,”, P+P). Platelets in treatment groups were suspended in HEPES buffer (0.2 M; pH 7.4; Fisher) at the same volume of Plasma that was removed. Platelets were then subjected to treatment with PARIAP (0.1 M, 0.01M), Thrombin (0.4, 4 U/mL; Sigma Aldrich), CaCl₂ (0.5wt.%; Fisher), or HEPES buffer (control) for 30 minutes at room temperature, which is consistent with previous literature^{15 (link)–17 (link)}. Platelet suspensions in freeze/thaw treatment group were subjected to freezing three times in liquid nitrogen (5 minutes) and thawing in a 37°C water bath (10 minutes). After activation, platelet suspensions were centrifuged at 2000 × g for 10 minutes, and the supernatant (hereafter referred to as “platelet concentrate,” PC) was sterile filtered through a 0.2 μm filter and collected for processing. Supernatants and controls were stored at -80°C before ELISA was performed as described below.

Belair D.G., Le N.N, & Murphy W.L. (2016). Regulating VEGF Signaling in Platelet Concentrates via Specific VEGF Sequestering. Biomaterials science, 4(5), 819-825.

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Outer Membrane Permeabilization Assay

Cited 1 time

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Disruption of the OM of bacteria was detected using the N-phenyl-1-napthylamine (NPN) uptake assay as reported previously [12 (link)]. Bacteria grown to stationary phase overnight were washed three times in MHB by centrifugation (12 300 g, 3 min) and resuspension. Washed bacteria were diluted to an OD_600nm of 0.5 in 5 mM HEPES buffer (Sigma-Aldrich, USA) and added to a black-walled microtitre plate. NPN (Acros Organics, USA) was diluted in HEPES buffer and added to the relevant wells to achieve a final concentration of 10 µM. Colistin was diluted in HEPES buffer and added to the relevant wells to achieve a final concentration of 4 μg ml⁻¹. Fluorescence was measured immediately using a Tecan Infinite M200 Pro microplate reader (Tecan Group, Switzerland) using an excitation wavelength of 355 nm and an emission wavelength of 405 nm. Measurements were obtained every 30 s for 1 h and all data averaged to give a mean fluorescence value. The degree of OM permeabilisation was calculated as the NPN uptake factor [28 (link)]:

\frac{F l u o r e s c e n c e o f s a m p l e w i t h N P N - F l u o r e s c e n c e o f s a m p l e w i t h o u t N P N}{F l u o r e s c e n c e o f H E P E S b u f f e r w i t h N P N - F l u o r e s c e n c e o f H E P E S b u f f e r w i t h o u t N P N}

Humphrey M., Larrouy-Maumus G.J., Furniss R.C., Mavridou D.A., Sabnis A, & Edwards A.M. (2021). Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic. Microbiology, 167(11), 001104.

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Outer Membrane Permeabilization Assay

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Disruption of the OM of bacteria was detected using the N-phenyl-1-napthylamine (NPN) uptake assay [12] . Bacteria grown to stationary phase overnight were washed three times in MHB by centrifugation (12, 300 x g, 3 minutes) and resuspension. Washed bacteria were diluted to an OD600nm of 0.5 in 5 mM HEPES buffer (Sigma-Aldrich, USA) and added to a black-walled microtitre plate. NPN (Acros Organics, USA) was diluted in HEPES buffer and added to the relevant wells to achieve a final concentration of 10 μM. Colistin was diluted in HEPES buffer and added to the relevant wells to achieve a final concentration of 4 μg mL -1 . Fluorescence was measured immediately using a Tecan Infinite M200 Pro microplate reader (Tecan Group Ltd., Switzerland) using an excitation wavelength of 355 nm and an emission wavelength of 405 nm. Measurements were obtained every 30 seconds for 1 hour and averaged to give mean fluorescence. The degree of OM permeabilisation was calculated as the NPN uptake factor [28] :
𝑭𝒍𝒖𝒐𝒓𝒆𝒔𝒄𝒆𝒏𝒄𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒊𝒕𝒉 𝑵𝑷𝑵 -𝑭𝒍𝒖𝒐𝒓𝒆𝒔𝒄𝒆𝒏𝒄𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒊𝒕𝒉𝒐𝒖𝒕 𝑵𝑷𝑵 𝑭𝒍𝒖𝒐𝒓𝒆𝒔𝒄𝒆𝒏𝒄𝒆 𝒐𝒇 𝑯𝑬𝑷𝑬𝑺 𝒃𝒖𝒇𝒇𝒆𝒓 𝒘𝒊𝒕𝒉 𝑵𝑷𝑵 -𝑭𝒍𝒖𝒐𝒓𝒆𝒔𝒄𝒆𝒏𝒄𝒆 𝒐𝒇 𝑯𝑬𝑷𝑬𝑺 𝒃𝒖𝒇𝒇𝒆𝒓 𝒘𝒊𝒕𝒉𝒐𝒖𝒕 𝑵𝑷𝑵

Humphrey M., Larrouy‐Maumus G., Furniss R.C.D., Mavridou D.A., Sabnis A., & Edwards A.M. (2021). Colistin resistance in Escherichia coli confers protection of the cytoplasmic but not outer membrane from the polymyxin antibiotic.

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Evaluating Osteoinductive Potential of Materials

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Primary rat mesenchymal stem cells (rMSCs) were used to evaluate the osteoinductive potential of the different materials, e.g. their capacity to induce the differentiation of undifferentiated stromal cells to the osteoblastic phenotype. The effect of the materials on osteoblastic cells was assessed using human osteoblast-like SaOS-2 cells (ATCC, USA).
Although SaOS-2 cells are an osteosarcoma cell line, their suitability as osteoblast cell model has been widely demonstrated. [12] [13] [14] SaOS-2 cells were maintained in McCoy's medium (Sigma-Aldrich) supplemented with 10% FBS, 2 mM L-glutamine, penicillin/ streptomycin (50 U/mL and 50 µg/mL respectively) and 20 mM HEPES buffer, all from Invitrogen. rMSCs were isolated from the tibias and femurs of Lewis rats at the Institute for Bioengineering of Catalonia (IBEC) as previously described. 15 The mesenchymal stem cell phenotype was previously characterized by flow cytometry. 16 Cells were expanded in Advanced DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) and 20 mM HEPES buffer, all from Invitrogen. Cells at passages 4-5 were used in all experiments.

Sadowska J.M., Guillem-Marti J., Montufar E.B., Espanol M, & Ginebra M.P. (2017). ^* Biomimetic Versus Sintered Calcium Phosphates: The In Vitro Behavior of Osteoblasts and Mesenchymal Stem Cells. Tissue engineering. Part A, 23(23-24).

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Isolation and Co-culture of Vaginal T Cells

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The resected vaginal tissue was digested in RPMI 1640 medium (GIBCO,
Grand Island, NY) containing collagenase-D (400 unit ml⁻¹,
Roche Indianapolis, IN), DNase-I (2.5mg ml⁻¹, Roch),
Pen-Strep (100 μg ml⁻¹, GIBCO), HEPES buffer (20 mM,
GIBCO) and fetal bovine serum (10%, GIBCO). The digestion lasted for 20
minutes with stirring. The vaginal tissue was then smashed through a 70
μm cell strainer (Fisher, Pittsburgh, PA) followed by CD8 depletion
using Dynal Beads (Life Technologies). The CD8-depleted vaginal tissue, either
with or without CD11c-depletion (CD11c kit, Miltenyi Biotec), was co-cultured
with 1×10⁶ CFSE (Invitrogen) labeled naïve OT-1
CD8⁺ T cells in RPMI medium containing Pen-Strep (100
μg ml⁻¹, GIBCO), HEPES buffer (10mM, GIBCO),
L-glutamine (2 mM, GIBCO), gentamicin (50mg ml⁻¹, SIGMA), MEM
non-essential amino acid (GIBCO), sodium pyruvate (1mM, GIBCO),
2-mercaptoethanol (10 μM, SIGMA), fungizone (2.5 μg
ml⁻¹, GIBCO) and fetal bovine serum (20%,
GIBCO).

Wang Y., Sui Y., Kato S., Hogg A.E., Steel J.C., Morris J.C, & Berzofsky J.A. (2015). Vaginal type-II mucosa is an inductive site for primary CD8+ T-cell mucosal immunity. Nature communications, 6, 6100.

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Isolation and Co-culture of Vaginal T Cells

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Isolation and Culture of Murine Neural Progenitor Cells

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NPSCs were isolated from the medial and lateral germinal eminences of E14.5 C57BL/6 mice based on previously published protocols [34 ] and in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Arizona State University. Briefly, mice were anesthetized at 3% isoflurane, rapidly decapitated, and fetuses were extracted from both uterine horns. Fetal tissue was rinsed in cold Leibovitz medium (Life Technologies, Carlsbad, CA) at each stage of the germinal eminence dissection. The germinal eminences were rinsed with sterile, cold Leibovitz medium before mechanical dissociation in working NPSC medium (glucose (6 mg/mL, Acros Organics, Geel, Belgium), HEPES buffer (5mM), progesterone (62.9 ng/mL), putrescine (9.6 µg/mL), heparin (1.83µg/mL), B27 growth supplement (1×, Life Technologies), epidermal growth factor (20 ng/mL), fibroblast growth factor (5 ng/mL), insulin (5 µg/mL), transferrin (5 µg/mL), sodium selenite (5 ng/mL) in Dulbecco's Modified Eagle Medium (Life Technologies), reagents from Sigma Aldrich unless otherwise specified) and plated at a density of 10⁴ cells/mL in a humidified incubator at 37°C, 20% O₂, and 5% CO₂. NPSCs were cultured as non-adherent neurospheres in working NPSC medium, passaged by mechanical dissociation, and utilized for experiments between passages 3 through 6.

Addington C., Heffernan J., Millar-Haskell C., Tucker E., Sirianni R, & Stabenfeldt S. (2015). Enhancing neural stem cell response to SDF-1α gradients through hyaluronic acid-laminin hydrogels. Biomaterials, 72, 11-19.

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Isolation and Haplotyping of Healthy Donor PBMCs

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PBMCs from healthy donors were isolated from leukocyte reduction filters purchased from the Rhode Island Blood Center (RIBC) in Providence, RI. High-resolution HLA Class II DRB1 haplotyping of donor PBMCs was performed at the Transplant Immunology Laboratory at Hartford Hospital in Hartford, CT. Donors’ age and sex are provided however race, ethnicity, and medical history are not available due to the anonymous nature of the blood donation process.
All assays were performed in RPMI complete medium: RPMI-1640 + GlutaMax (Life Technologies) containing 10mM HEPES buffer (Life Technologies), 2mM L-glutamine (Life Technologies), 50µg/ml Gentamicin (Life Technologies), 10% Human AB serum (Sigma), MEM Non-essential amino acids (Gibco) and 55µM β-Mercaptoethanol (Gibco).

De Groot A.S., Desai A.K., Lelias S., Miah S.M., Terry F.E., Khan S., Li C., Yi J.S., Ardito M., Martin W.D, & Kishnani P.S. (2021). Immune Tolerance-Adjusted Personalized Immunogenicity Prediction for Pompe Disease. Frontiers in Immunology, 12, 636731.

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