Cells from each cell line were plated on a cover glass-bottomed culture well at a density of 1 × 105 cells and incubated at 37 °C under 5% CO2 for 2 days. The cells were washed using Hanks’ balanced salt solution (HBSS, Wako) twice, and 1 μM SPiDER-βGal in HBSS was added to the cells and incubated for 15 min. Fluorescence images were captured using a fluorescence microscope (BZ-X810, KEYENCE, Japan). The TRITC filter set (excitation 545/25 nm, emission 605/70 nm, dichroic 565 nm) was used for fluorescence images; bright-field images were also acquired.
Hanks balanced salt solution (hbss)
Manufactured by Fujifilm
Sourced in Japan, United States
HBSS is a laboratory product manufactured by Fujifilm for use in cell culture and tissue research. It is a balanced salt solution that provides a physiologically compatible environment for maintaining cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Fujifilm (5 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Bz x810, by Keyence (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Dmem glutamax, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glass bottom dishes, by Matsunami (2 mentions)
Hanks balanced salt solution (hbss), by Keyence (2 mentions)
Bz x800, by Keyence (2 mentions)
Macs skeletal muscle dissociation kit for mouse and rat, by Miltenyi Biotec (2 mentions)
Satellite cell isolation kit, by Miltenyi Biotec (2 mentions)
Hoechst 33342, by Dojindo Laboratories (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Collagenase type 2, by Merck Group (2 mentions)
Facsmelody, by BD (1 mentions)
50 protocols using hanks balanced salt solution (hbss)
1
Quantifying Cellular β-Galactosidase Activity
2
Isolation and Characterization of Immune Cells
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The tissue of transplanted area was minced with a scissors, after which the tissue fragments were incubated for 1 h at 37 ºC in Dulbecco's Modified Eagle Medium (DMEM: Invitrogen) in the presence of 0.2% collagenase (Wako Chemicals, Cell dissociation grade), and 25 μg/ml deoxyribonuclease (Sigma-Aldrich). The suspension was filtered through a cell strainer (Falcon 2350, 70 μm) to remove debris and tissue fragments, after which the cells were pelleted by centrifugation at 300 g for 5 min at room temperature. Next, the cells were resuspended in calcium- and magnesium-free Hank's Balanced Salt Solution (HBSS) (Fujifilm) supplemented with 2% FBS, 10 mM HEPES and 1% penicillin/streptomycin (P/S). The cells were stained with immune cell markers as described below; PE conjugated mouse anti-rat CD3 (BD Pharmingen, Clone G4.18, Catalog No:554833), FITC conjugated mouse anti-rat CD4 (BD Pharmingen, Clone OX-35, Catalog No:554837), Catalog No:561588), PE conjugated mouse anti-rat CD11b/c (BD Pharmingen, Clone OX-42, Catalog No:554862), and Alexa Fluor® 647 conjugated mouse anti-rat CD163 (Bio-Rad Laboratories, Inc., Clone ED2, Catalog No: MCA342A647). Flow-cytometric analysis was performed on FACSMelody (BD Biosciences), and the data were analyzed using FlowJo software (BD Biosciences).
3
Reagents for Cell Culture and Protein Assays
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Reagents for tissue culture and transfection, DMEM (#SH30002, Cytiva, Marlborough, MA, USA), fetal bovine serum (FBS) (#175012, NICHIREI, Tokyo, Japan), Opti-MEM1 (#22600-134, Gibco, Billings, MT, USA), PEImax (#24885-2, Polysciences Inc., Warrington, PA, USA), and sodium 4-phenylbutyrate (4-PBA) (#O0511, TCI, Tokyo, Japan) were purchased. For SDS-PAGE and immunoblotting, mouse anti-His monoclonal antibody (#652501, Biolegend, San Diego, CA, USA), rat anti-FLAG monoclonal antibody (#637301, Biolegend, USA), HRP-anti-O-GlcNAc (#12938, CST, Danvers, MA, USA), CBB-R-250 (#031-17922, FUJIFILM, Tokyo, Japan), cell lysis buffer (10X) (#9803, CST, USA), and cOmplete protease inhibitor cocktail (#11697498001, Roche, Indianapolis, IN, USA) were purchased. For protein purification, Ni-NTA agarose (#143-09763, FUJIFILM, Tokyo, Japan), PreScission protease (#27-0843-01, Cytiva, Marlborough, MA, USA), Amicon filter (#UFC901096, Millipore, Burlington, MA, USA), empty polyprep chromatography columns (#7311550, Bio-Rad, Hercules, CA, USA), HBSS (#084-08965, FUJIFILM, Tokyo, Japan), and imidazole (#095-00015, FUJIFILM, Tokyo, Japan) were purchased.
4
Bovine Muscle Cell Isolation
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After dissection, the bovine muscle tissues underwent digestion with 2 mg/mL collagenase (FUJIFILM Wako Pure Chemical, Osaka, Japan), 10 mM HEPES (Life Technologies, Carlsbad, CA, USA), 1% penicillin/streptomycin (Life Technologies), and 10 μM DNase I (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle medium (DMEM)-GlutaMAX (Life Technologies) at 37 °C for 1 h with continuous shaking. Subsequently, the liquid fraction was collected, diluted two-fold using Hanks’ balanced salt solution (HBSS; FUJIFILM Wako Pure Chemical), and centrifuged at 800× g for 10 min at 20 °C. Then, the supernatant was removed. The cell pellet was resuspended in HBSS and filtered through a 100-μm cell strainer (Corning, Durham, NC, USA).
5
Measuring Cellular Oxidative Stress
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NHEKs or HaCaT cells were seeded in a 6- or 12-well plate at a density of 3 × 105 or 1.5 × 105 cells/well and incubated for 48 h. Cells were then washed twice with DPBS and treated with fresh medium containing DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BaP and BAI. Twelve hours later, cells were washed twice with Hank’s balanced salt solution (HBSS; Fujifilm Wako Pure Chemicals Corporation, Osaka, Japan) and stained with 25 μM H2DCFDA (Thermo Fisher Scientific) in HBSS for 30 min at 37 °C in the dark. After washing with DPBS, cells were harvested and used for flow cytometry. Mean fluorescent intensity (MFI) of DCF dye was analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA, USA). Propidium iodide (Thermo Fisher Scientific) was used to eliminate dead cells from the analysis.
6
Culturing Neuro-2A and Cerebellar Granule Cells
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Neuro-2A cells were cultured in DMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) at 37 °C under 5% CO₂. Cerebellar granule cells were extracted from 7-day-old ICR mice (Japan SLC, Inc., Shizuoka, Japan) in accordance with the Animal Care and Use Committee of Jichi Medical University and the ARRIVE guidelines, as described previously [15 (link)]. Briefly, mice were anesthetized with vaporized 30% isoflurane and then decapitated, and the cerebella were excised and treated with trypsin-EDTA (Thermo Fisher Scientific, Bothell, WA) for 7 min at room temperature. Dissociated cells were rinsed with HBSS (Wako Pure Chemical Industries, Osaka, Japan) three times and then plated into 4-compartment glass-bottom culture dishes (Greiner Bio-one, Kremsmünster, Austria) at a concentration of 4 × 10⁴/ml cells per well. Cells were kept in 0.5 ml Neurobasal Medium (Thermo Fisher Scientific) supplemented with B27 supplement (Thermo Fisher Scientific).
7
Fluorescent Lipid Uptake in Intestinal Intraepithelial Lymphocytes
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Total IELs were isolated from small intestine and washed twice with PBS. IELs (2 × 106) were suspended in buffer (2.5 mM CaCl2 and 1 mM MgCl2 containing HBSS from WAKO) and then incubated in buffer for 10 min at 37°C with 1.5 μM NBD-labeled PS (18:1–06:0 NBD-PS), PE (18:1–06:0 NBD-PE), or PC (18:1–06:0 NBD-PC) from Avanti Polar Lipid (Alabaster, AL, USA). After incubation, 5 mg/ml fatty acid–free BSA (SIGMA)-containing HBSS was added, and IELs were incubated for 5 min on ice. IELs were washed with fatty acid–free BSA containing HBSS. Flippase activity was assessed by measuring fluorescence in the IELs.
8
Isolation and Characterization of Pancreatic Cells
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Pancreatic tissues were carefully removed and immediately resuspended in 3 ml of Hank’s balanced salt solution (HBSS; Wako, Osaka) on ice. The tissues were cut into small pieces and digested in HBSS containing 2.5 mg/ml collagenase P and 0.1 mg/ml DNase I for 30 min at 37 °C. After incubation, the cells were washed with cold sterile phosphate-buffered saline (PBS) and collected by centrifugation at 1500 r.p.m. for 10 min. The cell pellets were resuspended in a cold HBSS medium, filtered through a 100-µm strainer (BD Falcon, no. 352360), and further collected by centrifugation at 1500 r.p.m. for 10 min. The cells were counted with a hemocytometer, and a Countess II FL automated cell counter (Invitrogen) was used. For edema analysis, whole pancreas samples were weighed and dried at 95 °C for 48 h. Edema was calculated following desiccation and is expressed as a ratio of the wet weight (wet weight − dry weight/wet weight × 100).
9
Isolation of Brain Immune Cells
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Brain immune cell isolation was performed as previously reported. Mice were deeply anesthetized using CO2, then perfused with 20 mL of ice‐cold HBSS (Wako). Brains were dissected into left (contralateral) and right (stroke) hemispheres. Each hemisphere was dissociated mechanically in RPMI 1640 (Wako) on ice. Immune cells were collected using the Percoll density gradient method. Finally, the collected cells were resuspended in 200 μL of stain buffer (fetal bovine serum) (BD Pharmingen) and used immediately for the fluorescence‐activated cell sorting.
10
Intracellular and Mitochondrial Iron Detection
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FerroOrange (Goryo Chemical Inc., Hokkaido, Japan) and Mito-FerroGreen (Dojindo) were used to detect intracellular and mitochondrial Fe2+. HeLa and SAS ρ0 cells were cultured overnight in glass-bottom dishes (Matsunami Glass). Then, the cells were washed twice with Hank's Balanced Salt Solution (HBSS) (Fujifilm Wako Pure Chemical Corporation) to remove residual FBS. The cells were treated with 1 μM FerroOrange or 5 μM Mito-FerroGreen in HBSS for 30 min at 37 °C. After incubation, FerroOrange and Mito-FerroGreen were removed by washing three times with HBSS. Fluorescence images were obtained using a BZ-8000 fluorescence microscope with GFP-BP and TRITC filters (excitation and absorption wavelengths: 540/25 and 605/55 nm). ImageJ software was used to measure fluorescence intensity.
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