Cell viability was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay. After treating RPE cells with A2E or BLL, MTT (40 μL/well, 0.4 mg/mL) was added and cells were incubated at 37 °C for an additional 40 min. The Formazan product was dissolved in 200 μL DMSO. Absorption was measured at 570 nm by using a microplate reader (MRX-TC; Dynex Technologies, Chantilly, VA, USA). Values were corrected for background absorbance by subtracting the appropriate blanks. Data are from at least six independent assays performed with three replicates each.
Mrx tc
Manufactured by Dynex
Sourced in Germany
The MRX-TC is a laboratory instrument designed for temperature control. It is capable of precisely regulating and monitoring the temperature of samples within a specified range.
Automatically generated - may contain errors
Lab products found in correlation
Superoxide dismutase activity colorimetric assay kit, by Abcam (1 mentions)
Hrp conjugated goat anti mouse igg, by Merck Group (1 mentions)
Nunc maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by BD (1 mentions)
Startingblock t20 blocking buffer, by Thermo Fisher Scientific (1 mentions)
2 n h2so4, by Carl Roth (1 mentions)
4 protocols using mrx tc
1
MTT Assay for Cell Viability
2
Assessing MnSOD2 Activity in VHL-deficient Cells
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MnSOD2 activity of conditioned media was analyzed using the Superoxide Dismutase Activity Colorimetric Assay Kit (Abcam, Cambridge, UK) according to the manufacturer's protocol using freshly prepared and undiluted 786-0VHL−- and 786-0VHL+-conditioned media. MnSOD2 activity was analyzed after incubation up to 60 min using a microplate reader (MRX-TC, DYNEX Technologies, Denkendorf, Germany). The absorbance values were expressed as percentage of the supernatant of 786-0VHL− cells.
3
Cell Viability Assay for Phospholipid Treatments
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HaCaT, J774A.1 cells and BMDCs were seeded at a density of 5 x 104 and 9 x 104 cells per well, respectively, onto 96-well flat-bottom plates and grown for 24 h at 37°C. Then, the medium was replaced by 100 μL of fresh medium with 5% FCS containing decreasing concentrations of empty UDA/UDL or dsLp- UDA/UDL in a half-fold dilution series (1.6 to 0.2 mg/ml of phospholipids, corresponding to 72 to 9 μg/ml of dsLp in UDL and 56 to 7 μg/ml of dsLp in UDA) or dsLp alone (200 to 50 μg/ml) and cells were incubated at 37°C for 24 h. After that, the medium was removed and replaced by 0.5 mg/ml of MTT. After 3 hours of incubation, the MTT solution was removed, the insoluble formazan crystals were dissolved in dimethyl sulfoxide, and absorbance was measured at 570 nm in a microplate reader (Dynex Technologies, MRX tc, Chantilly, Virginia). The cell viability was expressed as a percentage of the viability of cells grown in medium.
4
Mouse Antibody Titer Determination
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Antigen specific antibody-titer during mouse immunisation was determined by taking blood samples in two week-intervals starting from day 14. Serum from naïve mice served as preimmune control. ELISA assays were performed using 96 well Nunc MaxiSorp plates (Thermo Fisher Scientific, Germany) coated overnight with 500 ng/well respective antigen in coating buffer (15 mM Na2CO3, 35 mM NaHCO3 (pH 9.6)). After washing three times with 250 µl PBST, wells were incubated with 250 µl StartingBlock T20 blocking buffer (Thermo Fisher Scientific, Germany). Subsequently, serially diluted sera or pools of sera were added to individual wells in duplicates, and plates were incubated for 2 h at room temperature. Samples were then treated with HRP-conjugated goat anti-mouse IgG (Sigma Aldrich, Germany, cat. No. A3673) at final dilution of 1:3,000 for another 2 h at room temperature and afterwards developed with TMB-substrate (BD Biosciences, Germany). Reaction was stopped by addition of 50 µl 2 N H2SO4 (Carl Roth, Germany) and absorbance was measured at 450 nm using ELISA-reader (MRX TC, Dynex Technology, Germany or Multimode Reader TrisStar LB941, Berthold Technologies).
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