Spectramax paradigm multi mode microplate platform

HAP1 wild-type and KDELR1-KO cells (5 × 10⁴ cells each) were seeded in 96-well plates with phenol red-free DMEM medium (Gibco) supplemented with 10% FCS and 1% Pen/Strep and cultivated for 24 h. Subsequently, cells were treated with 1 or 1.5 µg/ml thapsigargin (Sigma Aldrich, in DMSO) or with the corresponding amount of DMSO in the negative control sample. After 24 h incubation, cell viability was determined via MTT assay. In brief, a solution of MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma) was added to a final concentration of 0.4 g/l (in 1 × PBS) and the 96-well plates were incubated for 3 h at 37 °C and 5% CO₂. Arosen crystals were dissolved by adding 100 µl solubilisation solution (Isopropanol 89.6%, HCl 0.4%, Triton X-100 10%) and slow shaking at 20 °C. Absorption was measured at 570 nm using „SpectraMax® Paradigm® Multi-Mode Microplate Platform“ (Molecular Devices) with the „Multi-Mode Analysis Software 2010“ (Molecular Devices). Calculation of mean values and standard deviations were performed with Microsoft Excel.

2 × 10⁴ cells of HAP1 wild-type and its KDELR1-KO variant were seeded in collagen-coated, laminin-coated or uncoated wells of a 96-well plate with IMDM medium, respectively. After adhesion for 4 h, unbound cells were removed by washing with 1 × PBS. Adherent cells were fixed with 2% paraformaldehyde (PFA, in 1 × PBS) for 15 min, washed twice with 1 × PBS and stained with 0.1% crystal violet solution (Merck) in 10% ethanol for 10 min. After 5 washing steps with sterile water, absorption was measured in a plate reader „SpectraMax® Paradigm® Multi-Mode Microplate Platform“ (Molecular Devices) with the „Multi-Mode Analysis Software 2010“ (Molecular Devices).