Cells were washed with PBS and lysed in a lysis buffer containing 50 mM HEPES (Promocell, Heidlberg, Germany), 1% Triton X-100, and protease and phosphatase inhibitors (Pierce Biotechnology, Rockford, IL, USA). Cellular protein concentration from total cell lysates (20 μg) was quantified using a bicinchoninic acid kit (Bio-Rad, Hercules, CA, USA). Samples containing equal amounts of protein were electrophoresed on 6%–8% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in TBST (in mmol/L, 10 Tris-HCl, 150 NaCl, 0.05% Tween-20, pH 7.6) and probed with the indicated primary antibodies (1:1,000) at 4°C overnight. Then the membranes were washed with TBST three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Billerica, MA, USA) for 1 h. The membranes were exposed to enhanced chemiluminescence kit according the manufacturer’s instructions (Beyotime Institute of Biotechnology). Image quantification was performed by ImageJ software (Version 1.41; NIH, Maryland, MD, USA).
Hepes
Manufactured by PromoCell
Sourced in Germany
HEPES is a chemical buffer solution used in cell culture and biochemical applications. It is designed to maintain a stable pH environment for biological systems. The core function of HEPES is to provide a buffering capacity that helps regulate the acidity or alkalinity of the solution, thus creating an optimal environment for cell growth and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Trypsin edta, by Capricorn (1 mentions)
Penicillin streptomycin, by PromoCell (1 mentions)
Penicillin streptomycin, by AppliChem (1 mentions)
Hepes, by GE Healthcare (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Anti tubulin antibody, by Abcam (1 mentions)
Anti phospho akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Phosstop, by Roche (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Completetm protease inhibitor cocktail, by Roche (1 mentions)
Streptomycin, by PromoCell (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
6 protocols using hepes
1
Western Blot Protein Analysis Protocol
2
Rheumatoid Arthritis Synovium Isolation
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Synovium of patients suffering from RA was obtained during knee replacement surgery (Agaplesion-Markus-Hospital, Frankfurt). All patients fulfilled the classification criteria of the American College of Rheumatology (13 (link)). This study was carried out in accordance with the recommendations of the ethic committee of the University of Giessen. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the ethic committee of the University of Giessen. Synovium samples were digested (1 h at room temperature, dispase-II-solution, 0.1 ml/ml, PAN-Biotech, Aidenbach, Germany) (14 (link)) and passed through cell strainers. After centrifugation, cells were cultured in DMEM (GE Healthcare, Germany) containing 10% heat-inactivated fetal calf serum (FCS, Sigma-Aldrich, Taufkirchen, Germany), 1 U/ml penicillin/streptomycin (AppliChem, Darmstadt, Germany), and 1 mM HEPES (GE Healthcare) at 37°C and 10% CO2 (14 (link)). Cells were passaged using trypsin/ EDTA (Capricorn, Ebsdorfergrund, Germany) for a maximum of passage 7 (15 (link)). Human umbilical vein endothelial cells (HUVEC) were cultured in Endothelial Cell Growth Medium 2 with recommended supplement mix (both PromoCell, Heidelberg, Germany) containing 1 U/ml penicillin/streptomycin and 1 mM HEPES at 37°C and 5% CO2.
3
Western Blot Analysis of Phosphorylated Akt
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ARVM and HL-1 cells were treated as indicated and lysed in buffer containing 50 mM HEPES (pH 7.4) (PromoCell, Heidelberg, Germany), 1 % Triton X-100 (Sigma-Aldrich, Munich, Germany), PhosSTOP and CompleteTM protease inhibitor cocktail (Roche, Basel, Switzerland). After incubation for 2 h at 4 °C, the suspension was centrifuged at 10,000g for 15 min. 5 μg protein sample of the total cell lysate was separated by SDS/PAGE (10 % gel) and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked in tris-buffered saline (TBS) containing 0.1 % tween 20 and 5 % (w/v) non-fat dried skimmed milk powder and incubated overnight with anti-phospho Akt(Ser473) antibody, anti-phospho Akt(Thr308), anti-GAPDH antibody (all Cell Signalling Technology, Danvers, MA, USA) or anti-tubulin antibody (Abcam, Cambridge, UK). After washing, membranes were incubated with appropriate horseradish peroxidase-coupled secondary antibody and processed for enhanced chemiluminescence (ECL) detection using Immobilion horse radish peroxidase (HRP) substrate (Millipore, Darmstadt, Germany). Signals were visualised and evaluated on a VersaDoc 4000 MP Bio-Rad Laboratories work station and analysed by Quantity One analysis software (version 4.6.7) (both Bio-Rad Laboratories, Hercules, CA, USA).
4
Isolation and Characterization of Human Mesenchymal Stem Cells
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HMADS cells used as human MSC model were isolated from adipose tissues obtained from young donors after informed parental consent as previously reported.44 (link) The human fetal ventricular RL14 cell line and primary HUVEC were purchased from the American Type Cell Culture (ATCC, LGC Standards S.a.r.l. Molsheim France) and PromoCell (Heidelberg, Germany), respectively.
HMADS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 1 g/l glucose, containing 10% heat-inactivated fetal bovine serum (FBS; Dominique Dutscher, Issy Les moulineaux, France), 100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM HEPES (Invitrogen, Carlsbad, CA, USA). As described earlier,44 (link) HMADS cells exhibited the following phenotype: CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1−, CD34−, CD15−, CD117−, Flk-1−, Gly-A−, CD133−, HLA-DR− and HLA-Ilow.
Human RL14 cells were grown in DMEM/F-12 (Life Technologies, Carlsbard, CA, USA) supplemented with 12.5% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM HEPES.45 (link) HUVEC cells were expanded on gelatin (2%)-coated dishes with the growth medium recommended and commercialized by the manufacturer (PromoCell). All cell types were maintained in a 5% CO2 atmosphere at 37 °C.
HMADS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), 1 g/l glucose, containing 10% heat-inactivated fetal bovine serum (FBS; Dominique Dutscher, Issy Les moulineaux, France), 100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM HEPES (Invitrogen, Carlsbad, CA, USA). As described earlier,44 (link) HMADS cells exhibited the following phenotype: CD44+, CD49b+, CD105+, CD90+, CD13+, Stro-1−, CD34−, CD15−, CD117−, Flk-1−, Gly-A−, CD133−, HLA-DR− and HLA-Ilow.
Human RL14 cells were grown in DMEM/F-12 (Life Technologies, Carlsbard, CA, USA) supplemented with 12.5% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 mM HEPES.45 (link) HUVEC cells were expanded on gelatin (2%)-coated dishes with the growth medium recommended and commercialized by the manufacturer (PromoCell). All cell types were maintained in a 5% CO2 atmosphere at 37 °C.
5
Murine Mammary Carcinoma Cell Culture
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The murine mammary carcinoma cell line 4T1 was obtained from ATCC (Manassas, Virginia, USA) and stably transfected with ptd‐Tomato‐N1 (Clontech, Saint‐Germain‐en‐Laye, France). Cells were cultured in RPMI‐1640 media (Thermo Fisher Scientific, Waltham, Massachusetts, USA) media, supplemented with 10 % FBS (Biochrom, Berlin, Germany) and 1 % HEPES (PromoCell, Heidelberg, Germany) at 37°C and 5% CO2. The murine endothelial cell line bEnd.3 and macrophage cell line RAW 264.7 were purchased from ATCC and cultured in DMEM (ATCC) supplemented with 10% FBS. Cells were routinely tested for mycoplasma contamination.
6
Western Blot Analysis of Signaling Pathways
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Cells were lysed in a lysis buffer composed of 50 mM HEPES (Promocell, Heidelberg, Germany), 1% TritonX100 (Sigma-Aldrich, Munich, Germany), complete protease inhibitor and PhosStop phosphatase inhibitor cocktail at pH 7.4 (Roche, Basel Switzerland). Protein lysates were diluted with Laemmli buffer and denatured for 5 min at 94°C. Five microgram protein were loaded on a 10% gel SDS-PAGE and transferred to a PVDF membrane during blotting process. Reagents for SDS-PAGE were supplied by Amersham Pharmacia Biotech (Braunschweig, Germany) and by Sigma-Aldrich (Munich, Germany). Blots were blocked in a Tris-buffered saline solution containing 0.1% Tween and 5% milk powder and incubated with antibodies against: PAR2 (SAM11: sc-13504, Santa Cruz Biotechnology, Heidelberg, Germany), COX2 (#4842), GAPDH (#2118), phospho-VEGFR2 (Tyr1059) (#3817), VEGFR2 (#2479), NF-κB p65 (#8242), and phospho-NF-κB p65 (Ser536) (#3033). Unless otherwise stated, all antibodies were purchased from Cell signaling Technology (Frankfurt, Germany). We used the corresponding secondary HRP-coupled antibodies against mouse and rabbit (Promega, Mannheim, Germany). After washing, blots were exposed to Immobilon HRP substrate (Millipore, Billerica, MA, USA) and analyzed with a VersaDoc 4000 MP work station (BIO-RAD, Munich, Germany).
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