We measured [Ca2+]i using the fluorescent Ca2+ indicator fura‐2‐acetoxymethyl ester (fura‐2/AM). Transfected HEK‐293 cells were loaded with fura‐2/AM to a final concentration of 1 μmol/L in complete medium and incubated at 37°C as reported previously (Ma et al. 2013 ). After 30‐min loading, the cells were washed three times with HEPES‐buffered saline (HBS) solution (145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 20 mmol/L HEPES‐NaOH, 2 mmol/L CaCl2, 20 mmol/L glucose, pH 7.4). The fluorescence of the cells loaded with fura‐2/AM was then measured at 37°C, at the determined sites, through a pinhole (10–20 μm in diameter). We used alternating excitation wavelengths of 340 and 380 nm in a Ca2+ microspectrofluorometric system (IX‐73 Model; Olympus, Tokyo, Japan) and Metafluor software (Molecular Devices, Sunnyvale, CA), as described previously (Hashii et al. 2000 ). The Ca2+ emission was detected every 5 sec for 5 min after application of PBS, OT, or analogs. The ratio of fluorescence at 340 nm and 380 nm (F340/F380) was used to determine [Ca2+]i. All data were normalized to the baseline fluorescence (F0) recorded 10 s before addition, and given as F/F0 where F – maximal ratio of fluorescence after drug's application. OT and analogs for experiments were diluted in 50% ethanol to a concentration of 10−3 mol/L and then diluted in distilled water to obtain the required concentrations.
Model ix73
Manufactured by Olympus
Sourced in Japan
The Olympus Model IX73 is an inverted microscope designed for a variety of applications in life science research. It features a robust and compact frame, providing a stable platform for high-quality imaging. The IX73 is equipped with a range of optical components, including infinity-corrected objectives, to deliver clear and uniform images across the field of view.
Automatically generated - may contain errors
Lab products found in correlation
Metafluor software, by Molecular Devices (1 mentions)
Costar 3464, by Corning (1 mentions)
Anti pycr1, by Proteintech (1 mentions)
Anti keratin, by Cell Signaling Technology (1 mentions)
Anti pro spc, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Axopatch 200b, by Molecular Devices (1 mentions)
9 protocols using model ix73
1
Measuring Intracellular Calcium Dynamics
2
Cell Migration Assay with U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In 24 well plates with transwell chamber inserts (Costar 3464, Corning), U2OS WT and MMP‐2 KO cells were seeded, separately, in the upper chamber at 20000 cells in 200 μL serum‐free DMEM. In the bottom chamber of the well 800 μL DMEM supplemented with 10% FBS was added. The plates containing triplicates of WT and KO were cultured for 24 h under standard culture conditions. The following day, non‐migrating cells were removed from the upper chamber and migrating cells were fixed with 70% ethanol (VWR, BT143215) and stained with 0.2% crystal violet (Millipore Sigma, V5265‐250ML). Membranes were left to dry and images were taken at 10× using Olympus microscope (IX73 model).
3
Rhodomyrtone-induced Nuclear Morphology
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW1353 cells were seeded in a 6-well plate at a density of 1.5 × 105 cells/well. After treatment with or without rhodomyrtone (0–5 μg/mL) for 24 h, the treated cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% paraformaldehyde, and stained with Hoechst 33342 for 15 min. The nuclear morphological changes were observed under a fluorescence microscope IX73 model (Olympus) with an ultraviolet filter.
4
Acyclovir-Resistant HSV-1 Mutant Generation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Previous literature has demonstrated that HSV isolates from cell culture are composed of a heterogeneous population, containing pre-existing antiviral-resistant thymidine kinase (TK) variants. Therefore, exposure of nucleoside analogues, such as acyclovir, in cell culture can provide selective pressure that can lead to enrichment of drug-resistant TK variants [33 (link)]. GFP-HSV-1 was used to generate an acyclovir-resistant (ACVR) mutant strain as previously described by Sarisky et al. (Figure 1 A) with the following changes: 70–80% confluent Vero cells in a 6-well plate were infected with 0.1 MOI of GFP-HSV-1 in the presence of 1 µg/mL ACV (Cat#2513, Tocris, Minneapolis, MN, USA). After observing a complete cytopathic effect (CPE), the virus was harvested and passaged onto fresh Vero cells (6-well plate) in the presence of 3 µg/mL ACV. The virus was harvested again after complete CPE and passaged for a third time in Vero cells with 10 µg/mL ACV. Plaques from the third passage were isolated, and the viral stock underwent three rounds of plaque purification in T:25 flasks. Fluorescence microscopy (Model IX73, Olympus, Tokyo, Japan) was used to image ACVR-HSV-1 using the FITC filter (excitation: 490 nm and emission: 525 nm).
5
Culturing and Characterizing CHO-K1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CHO-K1 cell line (Chinese hamster ovary cell line K1) was a generous gift from Drs. W. Trybus and E. Trybus (Department of Medical Biology, Institute of Biology, Jan Kochanowski University in Kielce, Poland). The CHO-K1 cells were cultured in DMEM/F12 1:1 containing 5% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 µg/mL amphotericin B in a humidified incubator (CO2 incubator HERAcell 150i, Thermo Electron LED GmbH, Langenselbold, Germany) at 37 °C and 5% CO2. The cultures were passaged twice a week using a 0.25% trypsin solution. The cells were observed using a phase-contrast microscope (Olympus, model IX73, Tokyo, Japan). All procedures necessitating sterile conditions were carried out using a laminar flow cabinet (Herasafe laminar flow cabinet, model KS, Thermo Electron LED GmbH, Langenselbold, Germany).
6
Visualizing Cellular Centrin5 Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells stably expressing TbCentrin5‐EYFP were harvested and washed twice with PBS. Resuspended cells were fixed with 4% paraformaldehyde and washed with PBS. Fixed cells were settled on the coverslip at room temperature for 30 min. The cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and examined with an inverted microscope (Model IX73; Olympus, Tokyo, Japan). Images were analyzed by imagej (NIH, Bethesda, MD, USA).
7
Immunofluorescence Staining of PYCR1, Keratins, Pro-SPC, and α-SMA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence, cells or slides were fixed (4% paraformaldehyde), permeabilized (0.5% Triton X-100), blocked (5% goat serum), and then stained with anti-PYCR1 (1:200, Proteintech, 13108-1-AP), anti-keratin (1:200, Cell Signaling Technology, 4545), anti–pro–surfactant protein C (anti–pro-SPC; 1:250, Abcam, ab90716), and anti–α-SMA (1:500, Servicebio, GB111364) primary antibodies overnight at 4°C. Afterward, samples were probed with secondary goat anti-rabbit or goat anti-mouse IgG antibody conjugated with Alexa Fluor 555 (Invitrogen, A-21428, A-21422) for 1 hour at room temperature in the dark. Nuclei were stained with DAPI (Yeason, 36308ES20). Images were visualized with a fluorescence microscope (Olympus, model IX73) and analyzed via ImageJ.
8
3T3-L1 Adipocyte Differentiation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3-Ll cells were cultured in DMEM supplemented with 10% CS and 1% P/S at 37°C in a 5% CO2 atmosphere. For 3T3-L1 cell differentiation, the cells when confluent were incubated for 2 more days (day 0) and then treated with MDI (500 µM IBMX, 0.25 µM dexamethasone, 10 µg/ml insulin) for 48 h at 37°C to induce differentiation. At 48 h after initiating 3T3-L1 cell differentiation, the DMEM medium (10% FBS, 1% P/S) was supplemented with 10 µg/ml insulin for 2 days (day 4). Day 4 after 3T3-L1 cell differentiation, DMEM medium (10% FBS, 1% P/S) was supplemented for 3 days (day 7). On day 7 of differentiation, differentiating cells were observed by capturing five different random fields of views for each well using a light microscopy at ×40 magnification (model IX73; Olympus Corporation). CsA (a CypB inhibitor) and mTOR-specific inhibitor rapamycin were dissolved in DMSO and added into the medium at 5 µg/ml (CsA) and 0.5 µM (Rapamycin), respectively at 37°C.
9
Patch-clamp setup for electrophysiological recordings
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The patch‐clamp setup was composed of a recoding chamber (Model RC‐24E, Warner instruments, Hamden, CT, USA) mounted on an inverted phase‐contrast microscope (Model IX73, Olympus Corporation, Tokyo, Japan) that was equipped with fluorescence visualization. The bath solution was applied by pump‐assisted perfusion using a syringe. Reference electrodes were made using bent glass capillaries that were mounted onto the recording chamber and contained 2.5 M KCl in 1% agarose. To isolate mechanical vibrations, the microscope was put on CleanBench laboratory table equipped with a Faraday cage (Technical Manufacturing Corporation, Peabody, MA, USA). The currents were amplified using a PC‐505B whole cell/patch‐clamp amplifier (Warner instruments, Hamden, CT, USA) or Axopatch 200b (Molecular Devices, San Jose, CA, USA), and acquired using Digidata 1322A/1550B and pClamp 9.2/11.2 software (Molecular Devices, San Jose, CA, USA). The signals were low‐pass filtered at 10 kHz and sampled at 20 kHz. The amplifier headstage was mounted on PatchStar MicroManipulator Controller (Scientifica Limited, East Sussex, UK). All voltage‐clamp protocols are described in the results section. All recordings were acquired at room temperature (24–26°C).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!