Rats were chosen for studies because they have a coronary anatomy that lends itself to generation of reproducible susceptibility to ischaemia-induced VF that is well characterised, and the in vitro (Langendorff perfusion) variation lends itself to the tractable evaluation of the concentration-dependence of the effects of drugs on VF and other variables40 (link). Male Wistar rats (254–475 g) were anaesthetised with a lethal dose of sodium pentobarbitone (170 mg.kg−1) and heparin (160 IU.kg−1) via intraperitoneal (i.p.) injection to achieve a surgical level of anaesthesia, confirmed by removal of pedal and corneal reflexes. Hearts were then excised (causing death by exsanguination) and immediately submerged in cold (4 °C) Krebs solution containing NaCl 118.5 mM, CaCl2 1.4 mM, glucose 11.1 mM, NaHCO3 25.0 mM, MgSO4 1.2 mM, NaH2PO4 1.2 mM and KCl 3 mM. Hearts were then cannulated via the aorta for Langendorff perfusion via the coronary arteries, and perfused with Krebs (as above), filtered (5μm pore size), warmed to 37 °C and gassed with 95% O2 and 5% CO2 to achieve a pH of 7.4, at a constant pressure of approximately 80 mmHg. Mexiletine (Sigma-Aldrich, UK) stock solutions were made up in a vehicle of 1:4, Ethanol (VWR International, UK): Water (PURELAB ELGA Process Water, UK). Krebs perfusate salts were purchased from VWR International, UK.
Krebs perfusate salts
Manufactured by Avantor
Sourced in United Kingdom
Krebs perfusate salts are a set of inorganic salts used to create a physiological buffer solution for organ and tissue perfusion experiments. The salts include sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium phosphate. This product provides the necessary ionic composition to maintain the appropriate osmotic pressure and pH for ex vivo studies.
Automatically generated - may contain errors
Lab products found in correlation
Phospho mkk7 ser271 thr275, by Cell Signaling Technology (2 mentions)
Hrp linked antibody, by Cell Signaling Technology (2 mentions)
Ambion micropoly a puris kit, by Thermo Fisher Scientific (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Sunitinib malate, by Merck Group (2 mentions)
Ethanol, by Avantor (1 mentions)
Mexiletine, by Merck Group (1 mentions)
Microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rno mir 1, by Thermo Fisher Scientific (1 mentions)
Hsa mir 27a, by Thermo Fisher Scientific (1 mentions)
Hsa mir 133a, by Thermo Fisher Scientific (1 mentions)
Hsa mir 133b, by Thermo Fisher Scientific (1 mentions)
Anti biotin hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ambion mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Total mkk7, by Cell Signaling Technology (1 mentions)
Phospho sapk jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Phospho ask1thr845, by Cell Signaling Technology (1 mentions)
Nqdi 1, by Merck Group (1 mentions)
3 protocols using krebs perfusate salts
1
Langendorff Perfusion of Rat Hearts
2
Sunitinib and miRNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Sunitinib malate and triphenyl-tetrazolium chloride were purchased from Sigma Aldrich (UK) and dissolved in dimethyl sulphoxide (DMSO) and stored at -20 °C. Krebs perfusate salts were from VWR International (UK) or Fisher Scientific (UK). Ambion MicroPoly(A)Puris kit, Ambion mirVana miRNA Isolation Kit, Reverse Transcription Kit, Applied Biosystems MicroRNA Reverse Transcription Kit, TaqMan Universal master mix II (no UNG), MKK7 mRNA primers, Applied Biosystems primers assays (U6, rno-miR-1, hsa-miR-27a, hsa-miR-133a, and hsa-miR-133b) were purchased from Life Technologies (USA).
The iTaq Universal SYBR Green Supermix was purchased from BioRad (UK). Phospho-MKK7 (Ser271/Thr275), Total MKK7 rabbit mAb antibodies and anti-rabbit IgG, HRPlinked antibody and anti-biotin, HRP-linked antibody were purchased from Cell signaling technologies (UK).
The iTaq Universal SYBR Green Supermix was purchased from BioRad (UK). Phospho-MKK7 (Ser271/Thr275), Total MKK7 rabbit mAb antibodies and anti-rabbit IgG, HRPlinked antibody and anti-biotin, HRP-linked antibody were purchased from Cell signaling technologies (UK).
3
Detailed Protocol for Sunitinib and NQDI-1 Signaling Pathway
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Sunitinib malate and NQDI-1 were purchased from Sigma Aldrich (UK). Both drugs were 140 dissolved in dimethyl sulphoxide (DMSO) and stored at -20 °C. Krebs perfusate salts were 141 from either VWR International (UK) or Fisher Scientific (UK). Total ASK1 (Catalogue no 142 ab131506) was purchased from Abcam (UK). Phospho-ASK1 (Thr 845) (Catalogue no 143 3765S), Phospho-MKK7 (Ser271/Thr275) (Catalogue no 4171S), Total MKK7 (Catalogue no 144 4172S), Phospho-SAPK/JNK (Thr183/Tyr185) (Catalogue no 9251), Total SAPK/JNK rabbit 145 mAb antibody (Catalogue no 9252), anti-rabbit IgG, HRP-linked antibody and anti-biotin, 146 HRP-linked antibody were purchased from Cell signalling technologies (UK). All the 147 primary antibodies were from a rabbit host, and MKK7 and JNK were monoclonal antibodies, 148 whereas ASK1 was polyclonal (all antibodies were validated by the manufacturers). The 149 Ambion MicroPoly(A)Puris kit, Ambion mirVana miRNA Isolation Kit and Reverse 150 Transcription Kit were from Life Technologies (USA). The mRNA primers and the Applied 151 Biosystems primers assays (U6, rno-miR-1, hsa-miR-27a, hsa-miR-133a, and hsa-miR-152 133b) were purchased from Invitrogen (UK).The iTaq Universal SYBR Green Supermix was 153 purchased from BioRad (UK). The HL60 cell line were obtained from European Collection of 154
Cell Culture (UK) (catalogue no. 98070106). 155 156
Cell Culture (UK) (catalogue no. 98070106). 155 156
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