Cells were plated in 6-well dishes (10000) in F12 medium or 24-well dishes (5000 or 7000) in assays conducted with viruses. Five days after transfection, or three after infection in the case of the virus tests, 0.63 mM of 3-(4,5-dimetilthyazol-2-yl)-2,5-dipheniltetrazolium bromide and 100 μM sodium succinate (both from Merck, Madrid, Spain) were added to the culture media and incubated for 2.5 h at 37 °C. After incubation, culture media were removed and the lysis solution (0.57% of acetic acid and 10% of sodium dodecyl sulfate (SDS) in dimethyl sulfoxide) (Merck, Madrid, Spain) was added. Absorbance was measured at 560 nm in a Modulus Microplate spectrophotometer (Turner BioSystems, Madrid, Spain). Cell viability results were expressed as the percentage of cell survival relative to the controls.
Modulus microplate spectrophotometer
Manufactured by Promega
Sourced in Spain, United States
The Modulus Microplate Spectrophotometer is a compact and versatile instrument designed for absorbance-based measurements in microplate format. It features a wide wavelength range and supports multiple detection modes to enable a variety of common assays and applications in the laboratory.
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Lab products found in correlation
4 protocols using modulus microplate spectrophotometer
1
MTT Assay for Cell Viability
2
Intestinal Barrier Function Assessment
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On day 10 -in the middle of the suckling period-, the functionality of the intestinal epithelial barrier was assessed on 3 rats of each litter (n = 12/group) by an in vivo permeability assay, as previously described (22 (link)). Briefly, a solution of 4 kDa-dextran conjugated to FITC (Sigma-Aldrich) was orally administered to rats using low-capacity syringes adapted to oral gavage tubes. There was an additional group of animals that was only administered with an equivalent volume of PBS (10 mL/kg) to rule out the fluorescent background of the plasma samples. After 4 h of the dextran administration, the animals were euthanized and plasma was obtained, diluted and the fluorescence emission was quantified by triplicate at an excitation wavelength of 490 nm in the Modulus™ microplate spectrophotometer (Turner Biosystems, Sunnyvale, CA, USA). Moreover, a 0.5 cm portion of the middle intestine was immediately conserved in RNAlater® (Applied Biosystems, Weiterstadt, Germany), incubated at 4°C overnight and stored at −20°C until PCR analysis.
3
MTT-Based Cell Viability Assay
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Cells (10,000) were plated in 6-well dishes in F12 medium or RPMI medium. Five days after transfection or 5-FU treatment, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sodium succinate (both from Sigma-Aldrich, Madrid, Spain) were added to the culture medium (final concentration 0.63 mM and 100 μM, respectively) and incubated for 2.5 h at 37 °C. Then, the culture medium was removed and the lysis solution (0.57% of acetic acid and 10% of SDS in DMSO) (Sigma-Aldrich, Madrid, Spain) was added. Absorbance was measured at 570 nm in a Modulus Microplate spectrophotometer (Turner BioSystems, Madrid, Spain). Cell viability results were expressed as the percentage of cell survival relative to the controls.
4
Intestinal Permeability Evaluation in Rats
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The evaluation of the intestinal epithelial barrier functionality was performed by an in vivo permeability assay to dextrans. Specifically, 4 kDa-dextran conjugated to FITC (Sigma-Aldrich, Madrid, Spain) in PBS at a dose of 500 mg/kg were orally administered to rats using low-capacity syringes (Hamilton Bonaduz, Bonaduz, Switzerland) adapted to oral 23-gauge gavage tubes (ASICO, Westmont, IL, USA), as previously described [23 (link)]. A reference sample pool obtained by animals given an equivalent volume of PBS was used to rule out autofluorescent background in each tissue sample itself.
The offspring were separated from their respective mothers 1 h before the administration, to allow gastric emptying and to avoid the interference of milk components with the dextran solution. Four hours after the administration, the animals were euthanized (ketamine (90 mg/kg)/xylazine (10 mg/kg)), plasma was obtained, diluted, and the fluorescence emission was analyzed by triplicate at an excitation wavelength of 490 nm, in the Modulus™ Microplate spectrophotometer (Turner Biosystems, Sunnyvale, CA, USA).
The offspring were separated from their respective mothers 1 h before the administration, to allow gastric emptying and to avoid the interference of milk components with the dextran solution. Four hours after the administration, the animals were euthanized (ketamine (90 mg/kg)/xylazine (10 mg/kg)), plasma was obtained, diluted, and the fluorescence emission was analyzed by triplicate at an excitation wavelength of 490 nm, in the Modulus™ Microplate spectrophotometer (Turner Biosystems, Sunnyvale, CA, USA).
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