All laboratory animal protocol was approved by the Institution of Animal Care and Use Committee of the Hualien Tzu Chi Hospital (IACUC Approval No. 108-10). Two-day-old neonatal Sprague-Dawley rats (both sexes) were used as cardiomyocyte donors and sacrificed by decapitation according to a previous report [25 (link)]. Briefly, the isolated hearts were washed using Hank's buffer (Gibco). The tissues were minced and digested with 0.045% pancreatin (Sigma) and 0.01% collagenase II (Gibco) in Hank's buffer, and the enzyme was then inactivated with culture medium (F-12 medium supplemented with 10% fetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin; Gibco). Cells were plated onto 10 cm petri dish and incubated for 1 h at 37°C in a fully humidified atmosphere with 5% CO2 incubator to reduce fibroblast contamination. The ventricular myocytes were collected and seeded on 0.1% collagen-coated 6 cm petri dish in culture medium with 10 μM cytosine arabinoside, and the medium was daily changed.
Hank s buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hank's buffer is a balanced salt solution commonly used in cell culture and other laboratory applications. It is designed to maintain the pH and osmotic balance of cell media, supporting the viability and growth of cells in vitro. The buffer contains a carefully formulated mixture of salts, glucose, and other components to create a physiologically relevant environment for cell culture.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Cytomics fc 500 mcl, by Beckman Coulter (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Collagenase p, by Roche (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Collagenase type 2, by Thermo Fisher Scientific (2 mentions)
R d kit systems, by R&D Systems (2 mentions)
Ethanol, by Merck Group (2 mentions)
Tween 80, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Lumicycler, by Actimetrics (2 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Collagenase 2, by Thermo Fisher Scientific (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Trypsin ethylenediaminetetraacetic acid, by Thermo Fisher Scientific (1 mentions)
Mouse insulin elisa kit, by Crystal Chem (1 mentions)
37 protocols using hank s buffer
1
Isolation and Culture of Neonatal Rat Cardiomyocytes
2
Transwell Flux Assay Protocol
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For flux assays, confluent monolayers in duplicate 6.5 mm Transwell filters were cultured in normal medium for 24 h, washed with pre-warmed Hank’s Buffer (Invitrogen 14025), and incubated in Hank’s Buffer for 30 min at 37 °C. The Buffer in the apical chamber was aspirated, and the filter transferred into a new well, containing 0.7 ml Hanks’ Buffer. 50 μl 3 kDa FITC-Dextran (Invitrogen D3305, stock 1 mg/ml) was added to the apical compartment. Cells were incubated at 37 °C for 2 h, and 0.1 ml of the basal compartment solution was removed, and replaced by 0.1 ml Hank’s Buffer. For calcium depletion experiments, after aspiration cells were rinsed 3X with calcium-free PBS, and incubated in calcium-free PBS, containing the tracer. Fluorescence of the basal compartment sample was measured with a spectrophotometer, and the amount of dextran (ng/ml) in each sample was determined by interpolation with a standard curve. The apparent permeability was calculated using the formula: Permapp = (ng/ml basal) x 106/Area well x Initial concentration x Time (sec).
3
Isolation and Culture of Murine DRG Neurons
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Inhalation anaesthesia was firstly applied to adult mice. Then dorsal root ganglia were dissected, rinsed with Hank’s buffer (Gibco, 13150016), and digested in the same buffer containing 1.5 mg/ml collagenase P (Roche Diagnostics, 11213865001) for 25–45 min at 37°C. Partially digested tissues were centrifuged at 200 × g for 3 min, and the pellets were resuspended in 0.25% trypsin-EDTA (Gibco, 25200056) and digested for an additional 5 min at 37°C. The digested ganglia were spun down, resuspended, and triturated with plastic pipette tips to release the neurons. The cells were filtered through a 70-μm cell strainer (Biologix, 15–1070), plated into 24-well plates, then aliquot and cultured in poly-d -lysine-treated 35 mm dishes containing DMEM/F-12 (Gibco, 11320033) supplemented with GlutaMAX (Gibco, 35050061) and 10% fetal bovine serum (Gibco, 10100). Electrophysiological experiments were performed after 2 h of culture.
4
Culturing Mouse Mammary and Melanoma Cells
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The mouse mammary adenocarcinoma TS/A cells [30 (link)], less immunogenic [31 (link),32 (link),33 (link),34 (link)] and mouse melanoma B16F10 more immunogenic cells (American Type Culture Collection, Manassas, Virginia, United States of America) [33 (link),35 (link)] were cultured in advanced minimum essential medium (AMEM, Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, United States of America) supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM/L L-glutamine (GlutaMAX, Gibco), 100 U/mL penicillin (Grünenthal, Aachen, Germany), and 50 mg/mL gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO2 humidified incubator at 37 °C. Cells of at least 80% confluence were trypsinized using 0.25% trypsin/ethylenediaminetetraacetic acid in Hank’s buffer (Gibco), washed with AMEM with FBS, collected by centrifugation (470 g, 5 min, Heraeus, ThermoFisher) and used in in vitro and in vivo experiments.
5
Pancreatic Insulin Content Measurement
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Islets were isolated using standard collagenase digestion followed by purification through a Ficoll gradient and islet gravity sedimentation. For total pancreatic insulin content, ten islets were handpicked, washed with Hank’s buffer (Gibco #14175), and lysed in 10 mM Tris-EDTA, 1% Triton-X 100, pH 8.0. Insulin content was measured using Mouse Insulin ELISA kit (Crystal Chem #90080).
6
Isolation and Culture of Murine DRG Neurons
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Newborn WT or TRPM8−/− mice were euthanized following the guidelines of the Animal Care and Use Committee of Kunming Institute of Zoology, Chinese Academy of Sciences. Dorsal root ganglia were dissected, rinsed with Hank's buffer (Gibco), and digested in the same solution containing 1 mg/ml collagenase P (Roche Diagnostics) for 15–30 min at 37°C. Partially digested tissues were centrifuged at 200 × g for 3 min, and the pellets were resuspended in 0.25% trypsin‐EDTA (Gibco) and digested for an additional 5 min at 37°C. The digested ganglia were spun down, resuspended, and triturated with plastic pipette tips to release the neurons. The cells were filtered through a 70‐μm cell strainer (Biologix), plated into 96‐well plates, and cultured in DMEM/F‐12 supplemented with GlutaMAX (Gibco), 10% serum (Gibco), and penicillin (100 U/ml)/streptomycin (0.1 mg/ml; Biological Industries). The calcium imaging experiment was performed after 48 h.
7
Glucose Transporter 1 Modulation Assay
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Transwell TM cell culture dishes were obtained from Corning Costar Corp (Cambridge, MA, USA). The CCK-8 kit was purchased from Beyotime (Shanghai, China). The MEM glucose medium, Hank’s buffer, and cell culture flask were obtained from Gibco (Grand Island, NY, USA). The Lucifer yellow was from Solarbio (Beijing, China). The MS-gradeacetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The formic acid (98.0%, for LC-MS) was purchased from Tokyo Chemical Industry (Shanghai, China). The glucose transporter 1 siRNA sequence: 5′-AUUCCAUUCACAGUGC UGAUCGCUC-3′; for the scrambled control siRNA: 5′-UACUCAG AACGAGUCUCGUUT-3′. The siRNA specifically targeting glucose transporter 1 and the control siRNA were purchased from Jikai Gene Chemical (Shanghai, China). The STF-31 was purchased from MCE (Shanghai, China). The resveratrol (purity greater than 99%) was purchased from Merck (Darmstadt, Germany).
8
Immunofluorescent Staining of Immune Cells
9
Flow Cytometry Analysis of Hepatocytes
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For flow cytometry analysis, the hepatocytes were obtained through shearing liver tissue, separating by collagenase type II (Thermo Fisher Scientific) digestion and suspended in RPMI 1640 medium (Thermo Fisher Scientific). Cell suspensions were centrifuged at 1,000 rpm for 5 minutes to remove cellular debris and impurities. Then, the hepatic mononuclear cells were harvested and resuspended in 70% percoll (Sigma). Mononuclear cells were collected from the interphase and washed twice in Hank’s buffer (Gibco, BRL). The experiments were performed according to the protocol of R&D kit systems (R&D Systems Inc.). Briefly, anti-CD4 FITC and anti-CD8 FITC antibody were added to the flow cytometry tube containing single-cell suspension, and these tubes were analyzed by Cytomics™ FC 500 MCL of Beckman Coulter, Inc (Sharon Hill, PA, USA).
10
Cell cycle detection
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HeLa and SiHa cells were digested with trypsin (Gibco; Thermo Fisher Scientific, Inc.) and prepared into a cell suspension. After cells were washed with Hanks buffer (Gibco; Thermo Fisher Scientific, Inc.), cells were centrifuged at 1,000 × g for 5 min at room temperature. Cells were then resuspended and incubated with pre-cooled 70% ethanol overnight. Finally, cells were stained with propidium iodide (30 µg/ml) for 30 min at room temperature (Beyotime Institute of Biotechnology), followed by cell cycle detection using a flow cytometer (BD LSRFFortessa; BD Biosciences). The results were analyzed using FlowJo software (version 10.6.2; BD Biosciences).
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