HREC proliferation was analyzed using Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) (32 (link)). The cells were diluted and seeded in a 96-well plate at a density of 8,000 cells/well in 100 µl of DMEM with 10% FBS and different concentrations of SDF-1α protein or SDF-1α protein plus CXCR4 antagonist. After incubation at 37°C for 24 h, 10 µl CCK-8 was added to each well and incubated at 37°C for 2 h in an incubator. Subsequently, absorbance was measured at a wavelength of 450 nm using a microplate reader (Thermo Multiskan EX plate reader; Thermo Fisher Scientific, Inc.).
Thermo multiskan ex plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Thermo Multiskan EX plate reader is a versatile microplate photometer that can be used for various absorbance-based assays. It offers a wide measurement range and can accommodate multiple plate formats. The device is designed to provide reliable and accurate results for a variety of applications in research and diagnostic laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Thiazolyl blue tetrazolium bromide, by Merck Group (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Heparan sulfate, by R&D Systems (1 mentions)
Streptomycin sulfate, by Thermo Fisher Scientific (1 mentions)
Costar tissue culture plates, by Corning (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Hexadecylpyridinium chloride monohydrate, by Merck Group (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Il 10, by BD (1 mentions)
Ifn α, by Mabtech (1 mentions)
Il 29, by R&D Systems (1 mentions)
Tnf α, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Keygen Biotech (1 mentions)
Horseradish peroxidase conjugated rabbit anti mouse igg, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using thermo multiskan ex plate reader
1
HREC Proliferation Analysis using CCK-8
2
Assessing MYPT1 Transfection Effect on Cell Proliferation
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SNU-5 cells (5×104 cells/mL) in the logarithmic phase were seeded into 96-well plates in 200 μL of medium and transfected with pcDNA3.1 and pcDNA3.1-MYPT1 for 48 h at 37°C with 5% CO2. To detect cell proliferation, 10 μL of Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added to each well. After 3 h, the absorbance was detected at 450 nm using a Thermo Multiskan Ex plate reader (Thermo Fisher, United Kingdom).
3
Cytotoxicity Assay of Salidroside and Imatinib
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Proliferations of THP-1 and U937 cells were investigated using the MTT assay as above. After the cells were seeded into 96-well plates, as above, and incubated overnight, the old RPMI-1640 media was removed and supplemented with an equivalent volume of FBS-free medium containing 2 mM salidroside, 20 μM imatinib, and 2 mM salidroside + 20 μM imatinib, respectively. The cells treated by the drug-free medium were used as the control. After 48 h of incubation, 20 μL MTT was added into each well and subsequently incubated with the cells for 4 h. Finally, the plates were subject to cell viability detection using a Thermo Multiskan EX plate reader (Thermo Fisher, United Kingdom) at a wavelength of 450 nm.
4
Murine BaF3 Cell Growth Assay
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Murine BaF3 lymphocytes expressing murine FGFR1IIIc were a gift from Professor David Ornitz (Washington University, St. Louis, USA). Cells were maintained in RPMI-1640 supplemented + 10% FCS + 100 Units/ml penicillin G + 100 μg/ml streptomycin sulfate (all from ThermoFisher, UK) and 1 ng/ml murine interleukin-3 (R&D Systems, UK) at 105 to 106 cells/ml at 37 °C, 5% CO2. For assays, cells were plated at 105 cells/ml in 96-well, flat bottomed Costar tissue culture plates (Corning, USA) in 100 μl growth medium without interleukin-3 supplemented with FGF-2 (R&D Systems, UK) and heparan sulfate at the indicated concentrations. Cells were incubated for 72 h at 37 °C, 5% CO2. Thiazolyl blue tetrazolium bromide (Sigma, UK) was then added to a final concentration of 250 μg/ml and the cells were incubated a further 4 h at 37 °C, 5% CO2. The assay was stopped with the addition of 50 μl 10% SDS, 0.01 N HCl and the plates were incubated 4 h to overnight at 37 °C to dissolve the formazan product. Plates were read at 570 nm using a Thermo multiskan EX plate reader (ThermoFisher, UK).
5
Quantification of Calcium Deposition
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Cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 minutes. Then, they were stained with 30 mM Alizarin Red S (pH 4.2, Sigma) for 10 minutes at room temperature. then, images were obtained with a Canon camera. In order to quantify calcium deposition, Mineralization of calcium nodules were quantified by a method described previously21 (link). Briefly, after staining, the cells were washed three times with PBS, 10% Hexadecylpyridinium Chloride Monohydrate (Sigma-Aldrich, Louis, MO, USA) was added and incubated for 20 mins at room temperature, then, Absorbance of the supernatant was measured at 540 nm in triplicate using ThermoMultiskan EX plate reader (Thermo Scientific, Waltham, MA, USA). Finally, the cells was washed with PBS and lysed with RIPA buffer and protein content was measured, the calcium levels were normalized to the total protein content.
6
Quantification of Cytokine Production
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The production of human GM-CSF, IFNα (both from Mabtech), IFNβ (PBL Interferon Source), IL-10, TNF-α, IFNγ (all from BD Pharmingen) and IL-29 (R&D Systems) in cell-free supernatant was determined using matched-paired antibodies according to the manufacturers’ instructions. Optical density absorbance readings were determined using a Thermo Multiskan EX plate reader (Thermo Fisher Scientific) at 405 nm absorbance.
7
Neutralizing Antibody Quantification Assay
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Neutralizing antibodies (NAb) were detected using a modified serial dilution assay of heat-inactivated (HI) patient serum as described previously (16 (link)). Briefly, 3-fold serial dilutions (1:3 to 1:531,441) of HI-serum samples were incubated with Pexa-Vec. After incubation for 3 hours at 37°C, the diluted HI-serum samples were transferred onto monolayers of Vero cells resulting in a final virus dose of 50 p.f.u./cell. Cells alone or with Pexa-Vec dilution only were also cultured as negative and positive controls, respectively. After a further 72 hours of incubation, MTT (5 mg/mL; Sigma) was added to each well and left for 4 hours, before the removal of all medium and addition of 150 μL DMSO (Thermo Fisher Scientific) to each well. Absorbance of samples was then read at 540 nm on a Thermo Multiskan EX plate reader (Thermo Fisher Scientific). NAb titers were calculated as 1/end point, which equates to the last serum dilution at which no antibody neutralization of Pexa-Vec–induced killing was observed (n = 9). Data are shown as mean + SEM.
8
Cell Viability Assay with MTT
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CHON-001 cells were seeded into 96-well plates at a density of 1 × 104 cells per well. After the above treatments, 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT; KeyGEN Biotech, Nanjing, China) was added to each well and the plates were incubated for 4 h in a 37 °C incubator. The medium was carefully removed from each well and the purple formazan was solubilized using dimethylsulphoxide (DMSO). The optical density at 590 nm (OD590) was determined using a Thermo Multiskan EX plate reader (Thermo Fisher Scientific).
9
Enzyme-Linked Immunosorbent Assay for Influenza Antibodies
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Sera were collected 2 wk after immunization to analyze antibody responses. Serum antibody titers against A-NP and B-NP were determined using ELISA. B/Yamagata/16/88 and A/PR/8 strains were split using 0.5% Triton X-100 (Sigma, St. Louis, MO, USA), and 96F Maxisorp immune plates were coated with the split viruses (3,600 PFU/well). The serum samples were diluted with 1% nonfat milk and 0.05% Tween 20/PBS and incubated at 25°C for 2 h. Thereafter, horseradish peroxidase-conjugated rabbit anti-mouse IgG (Abcam, Cambridge, UK) was added as a secondary antibody and incubated at 25°C for 1 h. Subsequently, 3,3′,5,5′-tetramethylbenzidine substrate and solution (KPL, Gaithersburg, MD, USA) were added to each well; thereafter, the reaction was terminated using 1 M phosphoric acid and detected at 450 nm using a Thermo Multiskan EX plate reader (Thermo Fisher Scientific, Vantaa, Finland).
ELISA results were expressed as endpoint titers. An endpoint titer is defined as the reciprocal of the highest analyte dilution that provides a reading above the cutoff. The cutoff formula used was as follows: “mean + 3 standard deviations of negative controls” (29 (link)).
ELISA results were expressed as endpoint titers. An endpoint titer is defined as the reciprocal of the highest analyte dilution that provides a reading above the cutoff. The cutoff formula used was as follows: “mean + 3 standard deviations of negative controls” (29 (link)).
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