For the epi-fluorescence imaging of ATTO594-NeuAcLc4Cer and ATTO594-Lc4Cer incorporated in cell plasma membranes (PMs), the fluorescent analogs were first dispersed in HBSS (Nissui), buffered with 2 mM PIPES (Dojindo) at pH 7.4 (P-HBSS) at a final concentration of 1.0 μM, and the dispersed solution was incubated with T24 cells on a glass-base dish (Iwaki) at 22 °C for 15 min. Then, the cells were chilled on ice-water (2.8 °C), washed three times with pre-chilled P-HBSS, and incubated in pre-chilled P-HBSS containing 1% (v/v) Triton X-100 on ice-water for 15 min.28 (link) Then, the cells were washed three times with cold P-HBSS, washed twice with pre-chilled PBS, fixed with 4% paraformaldehyde in PBS for 90 min, and observed under a Nikon Ts-2FL epi-fluorescence microscope equipped with a 60× 1.40 NA oil objective and a high-sensitive CMOS sensor-based camera (Nikon, DS-Qi2).
Hanks balanced salt solution (hbss)
Manufactured by Nikon
Sourced in Japan
HBSS is a balanced salt solution used in laboratory settings. It provides a physiological environment for cell culture and other applications requiring a balanced pH and ion concentration.
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Lab products found in correlation
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Ds qi2, by Nikon (1 mentions)
Hanks balanced salt solution (hbss), by Nissui Pharmaceutical (1 mentions)
Pipes, by Dojindo Laboratories (1 mentions)
Glass base dish, by Iwaki (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Smz745t, by Nikon (1 mentions)
Millicell cm, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Vt1000, by Leica (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
High vacuum grease, by Dow (1 mentions)
Image it live plasma membrane and nuclear labeling kit, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
5 protocols using hanks balanced salt solution (hbss)
1
Epi-fluorescence Imaging of Glycolipid Analogs
2
Measurement of Intracellular Oxidative Stress
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MEFs were cultured in 0.1% gelatin-coated 3-cm Petri dishes. After treatment with 0.1% DMSO for the indicated time, cells were washed with warm Hanks’ balanced salt solution (HBSS: Gibco-Thermo Fisher Scientific) and then loaded with 25-μM chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-H2DCFDA; C-6827, Life Technologies) in complete growth medium for 10 min at 37°C in the dark. After loading, cells were washed twice with HBSS and examined using a Nikon inverted fluorescence microscope. Three to five fields were selected for imaging with a 10× objective lens and quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). The value of WT basal (set as 1.0) was used to normalize the results.
3
Circadian Rhythm Monitoring in SCN and Kidneys
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Animals were anesthetized with isoflurane and sacrificed under dim light (43 ± 10 lux; mean ± SD) during their subjective daytime. Brains and kidneys were quickly removed and immersed in ice-cold HBSS (Gibco). Paired sets of the SCN and kidneys were isolated from the same animals. For imaging and luminometry, kidneys were sliced at 50–100 μm thickness on a vibratome (Leica VT1000S, Heidelberg, Germany). For luminometry, the whole SCN was carved out in ice-cold HBSS under a surgical microscope (Nikon SMZ745T, Tokyo, Japan) using cut blades as described previously [8 (link)]. Each explant was transferred to culture membrane insert (Millicell-CM, Millipore, Bedford, MA, USA) and cultured at 37 °C in a transparent 35-mm dish (Corning, Corning, NY, USA) or 35-mm glass-bottom dish (Alpha Plus Scientific, Taoyuan, Taiwan), with the lid sealed with High Vacuum Grease (Dow Corning, Midland, MI, USA). Explants were cultured in B27-supplemented Neurobasal-A (Gibco) medium containing: 4.2 mM sodium bicarbonate (Gibco), 10 mM HEPES (Gibco), 1% GlutaMAX (Gibco), 1% penicillin-streptomycin (10 U/μL penicillin and 10 μg/μL streptomycin, Gibco), 2% B-27 (Gibco), and 300 μM beetle luciferin (VivoGlo P1043, Promega, Madison, WI, USA), at pH 7.3 at 37 °C before addition of B-27 and antibiotics.
4
PLACC900 Exposure on BEAS-2B Cells
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BEAS-2B cells were seeded on glass coverslips (15 mm diameter; Fisher Scientific) in a 12 well plate at a density of 1.5 × 105 cells/ml overnight. The cells were subsequently exposed to 100, 300, or 500 µg/ml PLACC900 dispersed in media as previously described. After 24 h, the media was removed and the cells were washed two times with Hank’s Balanced Salt Solution (HBSS) (Corning, Inc.), fixed with 4% formaldehyde (Sigma-Aldrich) for 15 min and at 37 °C, and subsequently washed 3 more times with HBSS to remove any remaining formaldehyde. The cells’ plasma membranes and nuclei were then stained with 3 µg/ml Alexa Fluor 594 wheat germ agglutinin (WGA) and 2 µM Hoechst 33342 (Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit, Life Technologies), respectively, both dispersed in HBSS, for 10 min and at 4 °C. After incubation, cells were washed 2 times with HBSS, the cover slides were mounted on glass coverslips, and imaged under a Nikon Inverted Microscope Eclipse Ti Series (Nikon) and a 40X objective. The NIS-Elements BR 3.1 software was used to analyze the size and morphology of the cells.
5
Evaluating SkM Viability on HAM and EF-HAM Scaffolds
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The viability of SkM on HAM and EF-HAM scaffolds was assessed through Live/Dead assay which distinguishes live from dead cells through staining with Calcein AM (Cayman Chemical, Ann Arbor, MI, USA) and ethidium homodimer-1 (EthD-1; Thermo Fisher), respectively. SkM were seeded on scaffolds at a 10,000 cells/cm2 seeding density and cultured for 48 h before staining procedure. For staining, cells were incubated with a mix solution of 2 µM of Calcein AM and 4 µM of EthD-1 prepared in Hank’s 1X Balanced Salt Solutions (HBSS; Cytiva, Marlborough, MA, USA) at room temperature for 30 min. Staining solution was then replaced with HBSS and the images of stained cells were captured at five different fields using fluorescence microscope (Nikon, Tokyo, Japan) before the number of live and dead cells were counted and averaged.
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