Freshly harvested pancreatic and colonic tissues were digested in 1.0 mg/mL collagenase-P (Boehringer, Mannheim, Germany) solution at 37°C for 15 min and filtered through 75 µm filters with hank’s solution (Beyotime, Shanghai, China). Single-cell suspensions were incubated for 30 min at 4°C in hank’s solution with the following mAbs: APC Rat Anti-Mouse CD11b, BV421 Rat Anti-Mouse F4/80, Alexa Fluor 700 Rat Anti-Mouse Ly-6G, PE Hamster Anti-Mouse CD11c, FITC Rat Anti-Mouse MHCII, and PerCP-Cy5.5 Rat Anti-Mouse CD8a (BD Pharmingen, CA, USA). Gating method of fluorescence-activated cell sorting was programmed as CD11b+ Ly-6G+ (for neutrophils), CD11b+ F4/80+ (for macrophages), CD11c+ MHCII+ (for conventional dendritic cells, cDCs), and CD8a+CD11c+ MHCII+ (for plasmacytoid dendritic cells, pDCs). Flow cytometer was performed on Attune NxT (Thermo Fisher Scientific, MA, USA). Data were analyzed using ACEA NovoExpress software (Novo Express International, Inc., South San Francisco, CA, USA).
Hank s solution
Manufactured by Beyotime
Sourced in China
Hank's solution is a laboratory reagent used in various biochemical and cell biology applications. It is a balanced salt solution designed to maintain the osmotic and pH balance of cells and tissues during in vitro experiments.
Automatically generated - may contain errors
Lab products found in correlation
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Apc rat anti mouse cd11b, by BD (1 mentions)
Collagenase p, by Boehringer Ingelheim (1 mentions)
Bv421 rat anti mouse f4 80, by BD (1 mentions)
Pe hamster anti mouse cd11c, by BD (1 mentions)
Inverted microscope, by Nikon (1 mentions)
Selisistat, by Merck Group (1 mentions)
Exendin 9 39, by Merck Group (1 mentions)
5 protocols using hank s solution
1
Tissue Digestion and FACS Immunophenotyping
2
LNA Complexation Using BSA
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Since LNA, as a free fatty acid, is not water soluble, BSA was used to complex LNA as previously described [12 (link)]. Briefly, a stock solution of 200 mM LNA (purchased from Sigma-Aldrich, Saint Louis, MO, USA) was prepared in 96% EtOH and then diluted into 4 mM LNA in 10% BSA (Sigma-Aldrich)/Hanks’ solution (Beyotime, Nantong, China). The mixture was incubated at 37 °C, vortexing for about 30 min. Then, this solution was diluted in complete cell culture medium as 200 µM LNA (complexed to 0.5% BSA) for exposure. The complete cell culture medium containing 0.5% BSA and 0.1% EtOH was used for the control groups.
3
Islet Staining and Microscopic Imaging
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The islet suspension was transferred to a 15-ml centrifuge tube for centrifugation (300 × g for 3 min), and the supernatant was discarded after centrifugation. Then, the pellet was resuspended and transferred to a six-well plate with 2 ml Hank’s solution (Beyotime). About 20 µl of dithizone staining solution (0.67 mg/ml) (Solarbio, Beijing, China) was added in each well and incubated in dark for 10 min at room temperature, and then images were captured under an inverted microscope (Nikon, Japan).
4
Isolation and Propagation of DAdV-3
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The liver tissues of a 30-day-old DAdV-3 infected Muscovy duck were collected from the Avian Disease Diagnostic Center of Anhui Agricultural University, Hefei, China, and used as a DAdV-3 source for this study. The liver was cut into pieces and grounded into a homogenate mixture under sterile conditions. Subsequently, Hank's solution (Beyotime, Shanghai, China) was added to this homogenate mixture to make a 10% suspension. After this suspension was frozen and thawed 3 times, then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was filtered through a 0.22 μm filter and stored at −80°C for further use.
The supernatant was inoculated with the dose rate of 0.15 mL/embryo into the allantoic cavity of 8-day-old SPF chicken embryos. The inoculated embryos were incubated at 37°C with a relative humidity of 55%. The embryos that died within 2 d were discarded, whereas those that died between 9 and 10 d were stored for the collection of DAdV-3.
The supernatant was inoculated with the dose rate of 0.15 mL/embryo into the allantoic cavity of 8-day-old SPF chicken embryos. The inoculated embryos were incubated at 37°C with a relative humidity of 55%. The embryos that died within 2 d were discarded, whereas those that died between 9 and 10 d were stored for the collection of DAdV-3.
5
Hypoxia-Induced Cardiomyocyte Injury Model
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In vitro, chronic hypoxia of cardiomyocytes was used to mimic the infarcted heart following AMI. Hypoxic conditions were produced using fresh Hank's solution (Beyotime Institute of Biotechnology) with 95% N2 and 5% CO2. The dishes (1×106 cells) were placed into a hypoxic incubator at 37°C, that was equilibrated with 95% N2 and 5% CO2 and the actual oxygen concentration was 0. Ambient O2 levels in the hypoxic incubator were monitored with an O2 analyzer (series-2000; Alpha Omega Instruments, Lincoln, RI, USA). Chronic hypoxia of cardiomyocytes cell line H9C2 (purchased from the American Type Culture Collection, Manassas, VA, USA) was performed for ~48 h. For liraglutide treatment, H9C2 were treated with liraglutide (0–50 nM) for 48 h in the presence of hypoxia. To activate SIRT1, pretreatment with SRT1720 (SRT; 10 µM; Sigma-Aldrich; Merck KGaA) was performed for ~4 h. To inhibit the SIRT1, Selisistat (10 µM; Sigma-Aldrich; Merck KGaA) was used for 6 h. To suppress the role of liraglutide, Exendin 9–39 (Ex9-39; 10 nM; Sigma-Aldrich; Merck KGaA) was used to block the glucagon-like peptide 1 receptor (GLP1R) under hypoxic conditions.
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