Adenovirus-infected cells were grown for 24 and 48 h in GM. Cells at each time point were counted (cell counting kit-8 #CK04; Dojindo Laboratories, Kamimashiki-gun, Kumamoto, Japan) according to the manufacturer’s instructions. The indirect cell number was expressed with absolute absorbance at 450 nm (Multiskan MS; Life Technologies Inc.). The proliferation rate was calculated as the cell number at 48 h divided by the cell number at 24 h [26 (link)].
Multiskan ms
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
The Multiskan MS is a multi-mode microplate reader that can perform absorbance and luminescence measurements. It is designed for a variety of applications in clinical and research laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 ck04, by Dojindo Laboratories (1 mentions)
Protease and phosphatase inhibitors, by Nacalai Tesque (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8 assay, by Dojindo Laboratories (1 mentions)
Mab466, by R&D Systems (1 mentions)
1 step ultra tmb, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Cell counting kit 8 cck 8, by Sangon (1 mentions)
Dif50, by R&D Systems (1 mentions)
Cell counting kit 8 (cck8), by Beyotime (1 mentions)
16 protocols using multiskan ms
1
Adenovirus-infected Cell Proliferation
2
Muscle Homogenization and Protein Extraction
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The frozen EDL muscles were thawed and homogenized (1:9 w/v) in ice-cold buffer (50 mM Tris-HCl, 200 mM NaCl, 50 mM NaF, 0.3% NP-40, pH 8.0) with protease and phosphatase inhibitors (Nacalai Tesque Inc., Chukyo-ku, Kyoto, Japan). After the muscle homogenates were centrifuged at 1000 × g for 20 min at 4°C, the supernatant was collected. Total protein concentrations of the supernatant were estimated using BCA protein assay reagent (Life Technologies Japan Ltd., Minato-ku, Tokyo) with bovine serum albumin as a standard using a microplate reader (Multiskan MS; Life Technologies Inc.). A part of the supernatant was used directly for CS activity measurements. The remainder of the supernatant was denatured with Laemmli sample buffer at 95°C for 5 min for one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Insoluble sediments of the homogenate described above were re-suspended (1:99 w/v) using MyHC sample buffer (30% glycerol, 5% ß-3-mercapto-1,2-propanediol, 2.3% SDS, 0.05% bromophenol blue, 62.5 mM Tris-HCl, pH 6.8). Then they were denatured at 65°C for 15 min.
Insoluble sediments of the homogenate described above were re-suspended (1:99 w/v) using MyHC sample buffer (30% glycerol, 5% ß-3-mercapto-1,2-propanediol, 2.3% SDS, 0.05% bromophenol blue, 62.5 mM Tris-HCl, pH 6.8). Then they were denatured at 65°C for 15 min.
3
Mitochondrial Content Estimation in EDL Muscles
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To estimate the mitochondrial content [32 (link)] of EDL muscles as described earlier, the CS activity was measured as described in a report of our early study [42 (link)]. The reaction mixture contained 100 mM Tris, 0.07% Triton X-100, 0.1 mM Ellman’s reagent (DTNB), 0.098 mM acetyl CoA, and 0.5 mM oxaloacetic acid at pH 8.3, and an appropriate volume of the supernatant prepared above [43 (link)]. Each measurement was taken spectrophotometrically (412 nm) in triplicate using a microplate reader (Multiskan MS; Life Technologies Inc.). The close two values were used for analyses. The activity was expressed as the specific activity corrected with the protein concentration of the supernatant (mM·min-1·mg protein-1).
4
Sulforhodamine B Assay for Cell Viability
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In order to detect growth inhibition sulphoradamine B (SRB) assay that measures changes in total protein content was applied. Cells were seeded in 96-well plate (3000–5000 cells/well). Cells were grown in the presence of vehicle, AICAR, MTX, AICAR+MTX for the time indicated (MCF-7 cells– 6 days, SKBR-3 cells– 3 days, 4T1 cells– 2 days, SAOS cells– 6 days, WM35–6 days). Cells were checked in an invert microscope every day. At the end of the treatment, after fixation in situ by 50% trichloroacetic acid (TCA) cell were stained with sulforhodamine B (SRB) solution (0.4% in 1% acetic acid). Unbound dye was removed by washing with 1% acetic acid. Bound stain was solubilized with 10 mM TRIS base. Absorbance was read on an automated plate reader (Thermo Labsystems Multiskan MS) at 540 nm.
5
Colorimetric Assay for Cell Viability
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Cell viability was determined by the MTT-based colorimetric assay (3 - [4,5 – dimethyl thiazol -2 -yl] -2,5- diphenyl tetrazolium bromide), Sigma-Aldrich Corporation St Louis, MO, USA) modified by Liu and Schubert (1997 (link)). Neurons were cultured in 96-well plates until they reached 80% of confluence in a final volume of 100 μl of medium containing 10% FBS and without phenol red, and incubated at 37°C in 5% CO2. After kinetic infection, the mitochondrial activity was measured by the modified MTT assay (Mosmann, 1983 (link)). This assay involves determining mitochondrial dehydrogenase activity in intact cells by incubation for 4 h at 37°C with MTT (5 mg/ml MTT solution per well). After the incubation, the resulting complex was solubilized by the addition of an acidified ethanol solution, and the absorbance was read at 570 nm on a Microplate reader (Thermo, Multiskan MS), and the results were expressed as a percentage (%) of the control cells. The results were analyzed statistically using the non-parametric Student t-test.
6
Cell Proliferation Assay with CCK-8
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Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to measure cell proliferation ability. HCC-1954, 21MT1 and JimT1 cells (3×103 cells/well) were plated in 96-well culture plates for 72 h. The CCK-8 reagent was added to each well and incubated at 37°C for 1 h. Cell viability was assessed by testing the absorbance at 450 nm using Multiskan MS (Thermo Labsystems, Helsinki, Finland).
7
Cell Proliferation Assay Protocol
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For assessing cell proliferation we applied the sulphoradamine B (SRB) assay, or, as an alternative, BRDU-incorporation assay using the Cell Proliferation ELISA, Brdu (colorimetric) kit (Hoffmann-La Roche, Basel, Switzerland) according to the manufacturer’s instructions. In the SRB assay cells were seeded in 96-well plate (3000–5000 cells/well) and depleted of PARP10 using specific shRNA expressing constructs. At the end of the treatment cells were fixed in situ by 50% trichloroacetic acid (TCA) and subsequently stained with sulforhodamine B (SRB) solution (0.4% in 1% acetic acid). Unbound dye was removed by washing with 1% acetic acid. Bound stain was solubilized in 10 mM TRIS base. Absorbance was read on an automated plate reader (Thermo Labsystems Multiskan MS, Walthman, Massachusetts, USA) at 540 nm.
8
Microtiter-based Herbicide Immunoassay
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Microtiter
plates were coated with the appropriate amount of antibody in PBS
(optimized by checkerboard titration) for 1 h at 37 °C, blocked
with PBS–BSA 1% at 37 °C for 30 min, and washed three
times with PBS 0.05% Tween 20 (PBST). Wells were dispensed with 50
μL of the herbicide standards in PBS or water samples plus 50
μL of the optimized dilution of the nanopeptamer-HRP conjugate
(Supporting Information ) in PBST. After
1 h of incubation and washing, 100 μL of the peroxidase substrate
(0.4 mL of 6 mg/mL 3,3′,5,5′-tetramethylbenzidine in
DMSO, 0.1 mL of 1% H2O2, in 25 mL of 0.1 M citrate
acetate buffer, pH 5.5) was added to each well, and the reaction was
stopped after 10 min by addition of 50 μL of 2 N H2SO4. Absorbance was read at 450/650 nm in a microtiter
plate reader (Multiskan MS, Thermo Labsystems, Waltham, MA). For the
analysis of field samples, the reaction mix was supplemented with
20% of interference buffer (1 M Tris, 0.3 M NaCl, 0.3 M EDTA, 1% BSA,
pH 7.4).
plates were coated with the appropriate amount of antibody in PBS
(optimized by checkerboard titration) for 1 h at 37 °C, blocked
with PBS–BSA 1% at 37 °C for 30 min, and washed three
times with PBS 0.05% Tween 20 (PBST). Wells were dispensed with 50
μL of the herbicide standards in PBS or water samples plus 50
μL of the optimized dilution of the nanopeptamer-HRP conjugate
(
1 h of incubation and washing, 100 μL of the peroxidase substrate
(0.4 mL of 6 mg/mL 3,3′,5,5′-tetramethylbenzidine in
DMSO, 0.1 mL of 1% H2O2, in 25 mL of 0.1 M citrate
acetate buffer, pH 5.5) was added to each well, and the reaction was
stopped after 10 min by addition of 50 μL of 2 N H2SO4. Absorbance was read at 450/650 nm in a microtiter
plate reader (Multiskan MS, Thermo Labsystems, Waltham, MA). For the
analysis of field samples, the reaction mix was supplemented with
20% of interference buffer (1 M Tris, 0.3 M NaCl, 0.3 M EDTA, 1% BSA,
pH 7.4).
9
Cellular Proliferation Assays for Cancer Cells
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Cellular proliferation was assessed using Sulphorhodamine B (SRB) and colony forming assays as described in Miko et al. and Fodor et al. [16 (link),97 (link)]. Cells were seeded in 96-well plates (4T1, 1500 cells/well; MDA-MB-231, 3000 cells/well; SKBR-3, 5000 cells/well; MCF7, 4000 cells/well; ZR75-1, 3000 cells/well; human fibroblast, 7500 cells/well) in complete medium and were cultured with different concentrations of IS for 24 h. Then, cells were fixed by the addition of 50% trichloroacetic acid (TCA, final concentration: 10%) and the plates were incubated for 1 h at 4 °C. Plates were washed five times in water and stained with 0.4% (w/v) SRB solution in 1% acetic acid. Unbound dye was removed by washing five times with 1% acetic acid. Bound stain was solubilized with 10 mM Tris base and the absorbance was measured on an automated plate reader (Thermo Labsystems Multiskan MS, Walthman, MA, USA) at 540 nm.
For colony forming assays, cells were seeded in six-well plates (4T1, 500 cells/well) and treated with the indicated concentrations of IS for seven days. After treatment, the plates were washed twice with PBS. Colonies were fixed in methanol for 15 min, dried, and stained with the solution of May-Grünwald-Giemsa for 20 min. Plates were washed with water and the colonies were counted using Image J software [98 (link)].
For colony forming assays, cells were seeded in six-well plates (4T1, 500 cells/well) and treated with the indicated concentrations of IS for seven days. After treatment, the plates were washed twice with PBS. Colonies were fixed in methanol for 15 min, dried, and stained with the solution of May-Grünwald-Giemsa for 20 min. Plates were washed with water and the colonies were counted using Image J software [98 (link)].
10
ELISA-based Detection of Chemokines
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Example 9
For detection of mCXCL10(1-77), Maxisorp Plates® (Nunc) were coated with 4 μg/ml of α-mCXCL10 capturing antibody (MAB466, R&D) in PBS and incubated over night at 4° C. Plates were washed twice with 300□l of PBS. Blocking was done with BSA 1% (proteinase free, Gibco), in PBS for 2 h at room temperature. Plates were washed 3 times with 300 μl of 0.05% Tween-20 in PBS. Tumor and plasma samples were diluted in BSA 1% and incubated for 2 h, at room temperature. To obtain a standard curve, and to control for the cross-reactivity of the detection antibody, dilutions of recombinant mCXCL10 (Peprotech) or DPP4-digested mCXCL10(3-77) were incubated in parallel. For detection of mCXCL10(1-77), biotinylated α-mCXCL10(1-77) (AbDSerotec, 0.5 μg/ml, clone AbD17185.1), Streptavidin-HRP (BD Biosciences) and 1-Step Ultra TMB (Thermo Scientific) were used. Enzymatic reactions were stopped with HCL 1N and plates were read with 450 nm in a Lab-systems Multiskan MS (Thermo) reader. For detection of total mCXCL10, the mCXCL10 DuoSet® ELISA kit (R&D) or a combination of capturing α-mCXCL10 (MAB466) and biotinylated α-mCXCL10 (BAF466, both from R&D) were used, unless otherwise indicated. Detection of mDPP4, mCCL22 and mCXCL12 was done with the DuoSet® ELISA kit (R&D). Detection of mVEGF, mCCL2 and mCCL3 was done with a multiplex kit (Invitrogen).
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