NaCl, KCl, NaH2PO4, KH2PO4, MgCl2, CaCl2 and glucose were purchased from Ajax-Finechem (Australia). HEPES was obtained from PanReac AppliChem (Darmstadt, Germany). Acetylcholine (ACh), dimethyl sulfoxide (DMSO), ethylene glycoltetraacetic acid (EGTA), nifedipine, tetraethylammonium chloride (TEA), methylene blue and Nω-nitro-l -arginine methyl ester (l -NAME) were purchased from Sigma (St. Louis, MO, USA).
Hepes
Manufactured by ITW Reagents
Sourced in Germany, Spain
HEPES is a widely used buffering agent in cell culture and biochemical applications. It maintains a stable pH range between 6.8 and 8.2, making it suitable for a variety of experimental conditions. HEPES is a zwitterionic organic compound that effectively buffers solutions to support optimal cellular and enzymatic activity.
Automatically generated - may contain errors
Lab products found in correlation
Tween 20, by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Cacl2, by Merck Group (3 mentions)
Sodium chloride (nacl), by ITW Reagents (2 mentions)
Sodium dodecyl sulfate (sds), by ITW Reagents (2 mentions)
Nifedipine, by Merck Group (1 mentions)
Tetraethylammonium chloride tea, by Merck Group (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Kh2po4, by Thermo Fisher Scientific (1 mentions)
Cacl2, by Thermo Fisher Scientific (1 mentions)
Methylene blue, by Merck Group (1 mentions)
Nω nitro l arginine methyl ester l name, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by ITW Reagents (1 mentions)
Buffer reagents, by Merck Group (1 mentions)
Caspase 3 substrate, by BD (1 mentions)
8 protocols using hepes
1
Vascular Smooth Muscle Pharmacology
2
SARS-CoV-2 Virus Production in Vero E6 Cells
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Vero E6 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Merck) supplemented with 1 mM sodium pyruvate (Merck), 1% nonessential amino acids (Merck), 4 mM l- glutamine (Merck), 50 μg/mL gentamicin (PanReac), 0.2 μg/mL antifungal (Sigma), and 10% fetal bovine serum (FBS; Sigma). Cells were cultured at 37°C and 5% CO2, and they were periodically thawed from a large frozen stock and passaged a maximum of 30 times at a split ratio of 1:6 to 1:8.
The virus used in the experiments was USA-WA1/2020, NR-52281 (deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH) (SRA accession no.NR-52281_70036318 ). To prepare a virus stock, 3 × 106 Vero E6 cells were infected with the virus at a multiplicity of infection (MOI) of 0.001 PFU/cell in DMEM supplemented with 25 mM HEPES (PanReac) and 2% FBS, and the infection was allowed to proceed for 48 h at 37°C. The titer of the viral stock was 1 × 107 PFU/mL. Virus infections were performed following standard procedures using closed flasks. To control for the absence of contamination, the supernatants of mock-infected cells, which were maintained in parallel with the infected cultures, were titrated; no infectivity in the mock-infected cultures was detected in any of the experiments.
The virus used in the experiments was USA-WA1/2020, NR-52281 (deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH) (SRA accession no.
3
Mouse HGF and Human BMP9 Assay Protocol
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Mouse recombinant HGF and human recombinant BMP9 were purchased from R&D Systems (Minneapolis, MN, USA). p38α/β MAPK inhibitor SB203580 was from Calbiochem (La Jolla, CA, USA). TGF-β activated kinase (TAK)1 inhibitor (5Z)-7-oxozeaenol was from Tocris Bioscience (Bristol, UK). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA were from Gibco-Invitrogen (Barcelona, Spain). Penicillin, streptomycin, propidium iodide, DNA oligos, and buffer reagents were from Sigma-Aldrich (Tres Cantos, Madrid, Spain). HEPES and bovine serum albumin (fraction V, fatty-acid free) from Panreac AppliChem (Castellar del Valles, Barcelona, Spain). Nucleospin RNA kit (Macherey-Nagel) from Cultek (Madrid, Spain). SuperScript III RNase H Reverse Transcriptase was from Invitrogen. Oligo-dT was from Roche Diagnostics (Sant Cugat del Valles, Barcelona, Spain). ECL reagent is from Thermo-Fisher Scientific (Madrid, Spain). Caspase-3 substrate was obtained from PharMingen (San Diego, CA, USA). Primary antibodies used in this study are listed in Table S2 .
4
Purification and Characterization of S100P Mutants
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Human S100P protein and its F89A and Δ42–47 (lacks PGFLQS sequence) mutants were prepared in E. coli as previously described [28 (link)]. The cytokine samples used in the present work are listed in Table 2 . Protein concentrations were measured spectrophotometrically using extinction coefficients at 280 nm calculated according to ref. [45 (link)].
Sodium acetate and ethanolamine were bought from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) HEPES, sodium chloride, and SDS were from PanReac AppliChem (Barcelona, Spain). Potassium hydroxide, CaCl2, EDTA, and TWEEN 20 were purchased from Sigma–Aldrich Co., (Burlington, MA, USA). Tricine was from Helicon (Moscow, Russia). Glutaraldehyde was from Amersham Biosciences (Little Chalfont, UK). Coomassie brilliant blue R-250 was bought from Merck (Darmstadt, Germany).
Sodium acetate and ethanolamine were bought from Bio-Rad Laboratories, Inc. (Hercules, CA, USA) HEPES, sodium chloride, and SDS were from PanReac AppliChem (Barcelona, Spain). Potassium hydroxide, CaCl2, EDTA, and TWEEN 20 were purchased from Sigma–Aldrich Co., (Burlington, MA, USA). Tricine was from Helicon (Moscow, Russia). Glutaraldehyde was from Amersham Biosciences (Little Chalfont, UK). Coomassie brilliant blue R-250 was bought from Merck (Darmstadt, Germany).
5
Recombinant Protein Characterization Protocol
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Human interferon beta-1a produced in CHO-K1 cells (IFN-β) was purchased from Merck (Rebif®). Human S100B was expressed in E. coli and purified as described in ref. [62 (link)]. Protein concentrations were measured spectrophotometrically according to ref. [64 (link)].
Sodium acetate, HEPES, NaOH, DTT and SDS were from PanReac AppliChem. Sodium chloride was from Helicon (Moscow, Russia). CaCl2, EDTA and TWEEN 20 were purchased from Sigma-Aldrich Co. NAP-5 column was from Cytiva.
ProteOn™ GLH sensor chip, amine coupling kit, EDAC and sulfo-NHS were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
MCF-7 cell line was from European Collection of Authenticated Cell Cultures. Crystal violet was from Sigma-Aldrich Co.
Sodium acetate, HEPES, NaOH, DTT and SDS were from PanReac AppliChem. Sodium chloride was from Helicon (Moscow, Russia). CaCl2, EDTA and TWEEN 20 were purchased from Sigma-Aldrich Co. NAP-5 column was from Cytiva.
ProteOn™ GLH sensor chip, amine coupling kit, EDAC and sulfo-NHS were from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).
MCF-7 cell line was from European Collection of Authenticated Cell Cultures. Crystal violet was from Sigma-Aldrich Co.
6
Recombinant Human S100A6 Protein Production
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The human S100A6 protein was expressed in E. coli, and purified as described in [44 (link)]. The samples of human cytokines are presented in Table S1 . The protein concentrations were determined according to [57 (link)].
The HEPES and sodium chloride were from PanReac AppliChem (Darmstadt, Germany). The CaCl2, Tween 20, and EDTA were purchased from Sigma Aldrich Co. (Burlington, MA, USA).
The HEPES and sodium chloride were from PanReac AppliChem (Darmstadt, Germany). The CaCl2, Tween 20, and EDTA were purchased from Sigma Aldrich Co. (Burlington, MA, USA).
7
Differentiation of LHCN-M2 Myoblasts
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LHCN-M2 myoblasts were purchased from Evercyte (Cat. No. CkHT-040-231-2) and cultured at 37 °C and 5% CO2 in a humidified incubator. The growth medium is based on the protocol from the manufacturer which consisted of four parts of Dulbecco’s Modified Eagle’s Medium (DMEM, 4.5 g/L glucose, Gibco) and one part of Medium 199 (M199) (Gibco), supplemented with 15% Fetal Bovine serum (FBS) (Gibco), 20 mM HEPES (PanReac Applichem), 0.03 µg/mL Zinc sulfate (Sigma-Aldrich), 1.4 µg/mL Vitamin B12 (Sigma-Aldrich), 0.55 µg/mL Dexamethasone (Sigma-Aldrich), 2.5 ng/mL HGF (Millipore), 10 ng/mL bFGF (Thermo Fisher Scientific) and 100 U/mL penicillin/streptomycin (Gibco).
To induce myotubes differentiation following the protocol previously described in ref. 50 (link), myoblasts were cultured until they reached a 50–70% confluency, followed by a change to differentiation medium consisting on DMEM (4.5 g/L glucose, Gibco)), supplemented with 2% Horse serum (Brunschwig), 10 µg/mL human insulin (Sigma-Aldrich), 5 µg/mL Holo-transferrin (Sigma-Aldrich), 10 nM sodium selenite (Sigma-Aldrich) and 100 U/mL penicillin/streptomycin. The cells were cultured at 37 °C and 5% CO2 in a humidified incubator for 5–7 days.
To induce myotubes differentiation following the protocol previously described in ref. 50 (link), myoblasts were cultured until they reached a 50–70% confluency, followed by a change to differentiation medium consisting on DMEM (4.5 g/L glucose, Gibco)), supplemented with 2% Horse serum (Brunschwig), 10 µg/mL human insulin (Sigma-Aldrich), 5 µg/mL Holo-transferrin (Sigma-Aldrich), 10 nM sodium selenite (Sigma-Aldrich) and 100 U/mL penicillin/streptomycin. The cells were cultured at 37 °C and 5% CO2 in a humidified incubator for 5–7 days.
8
Cryoprotectant Solution Composition and Characterization
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Cryoprotectant solutions (CSs) of the TK protocol (CS-TK) consisted of a mixture of Medium 199 1X (M199; Gibco, Madrid, Spain), a perfusion solution of 20% human albumin (Albutein 20%; Grifols, Barcelona, Spain), and dimethyl sulfoxide (DMSO; Sigma, Madrid, Spain); for AV protocol, they consisted of M199 and DMSO; for VS55 protocol, they consisted of Euro-Collins 5X [15 (link)], propylene glycol (Sigma, Madrid, Spain), DMSO, formamide (Sigma, Madrid Spain), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Panreac, Barcelona, Spain), and distilled water; for DP6 protocol, they included Euro-Collins 5X, propylene glycol, DMSO, and HEPES. The concentrations of the components of the CSs are shown in Table 2 . The CS were maintained at 4 °C for at least 1 h before use.
For each CS of TK protocol, the osmolality was measured in triplicate with a cryoscopic osmometer (Osmomat 030; Gonotec, Berlin, Germany) and the pH of each solution was determined using the pH-meter GLP21 (Crison, Castellón, Spain). Statistical analysis was performed with GraphPad Prisma software using a non-parametric test (Kruskal–Wallis test) to compare osmolalities among the different solutions, considering significant a p-value < 0.05. Results were expressed as the mean ± standard deviation.
For each CS of TK protocol, the osmolality was measured in triplicate with a cryoscopic osmometer (Osmomat 030; Gonotec, Berlin, Germany) and the pH of each solution was determined using the pH-meter GLP21 (Crison, Castellón, Spain). Statistical analysis was performed with GraphPad Prisma software using a non-parametric test (Kruskal–Wallis test) to compare osmolalities among the different solutions, considering significant a p-value < 0.05. Results were expressed as the mean ± standard deviation.
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