Calpain activity fluorometric assay kit

Calpain activity was measured with the Calpain Activity Fluorometric Assay Kit (Sigma Aldrich, St. Louis, MO, United States), following manufacturer protocol. Samples were measured in triplicates. In short, the amount of 2 × 10⁶ erythroblasts was taken per sample. Samples were incubated with or without Yoda1 (1 μM or 5 μM, 10 min, 37°C). After centrifugation, samples were washed in PBS and resuspended into 100 μl Extraction Buffer. Samples were incubated (20 min, on ice) and spun down (10.000 g, 1 min). Cell lysate was transferred to a new tube and kept on ice. 10x Reaction buffer and Calpain substrate was added to 85 μl cell lysate and samples were transferred to 96-well plate. After incubation in the dark (1 h, 37°C), samples were measured with 400 nm excitation filter and 505 nm emission filter with a plate reader (BioTek, Winooski, VT, United States). Positive control (1 μl Active Calpain) and negative control (1 μl Calpain inhibitor) were taken along. Data was analyzed with Prism 8. Two-way ANOVA was used to calculate statistical significance between samples.

To confirm the inhibitory effect of Calp1 we used a commercially available Calpain Activity Fluorometric Assay Kit (Sigma-Aldrich, #MAK228). The assay reaction was prepared by following the manufacturer’s instruction except for adding CALP1 either alone or to the Active Calpain (Sigma-Aldrich, #MAK228D) in a final concentration of 10 µM. After one hour of incubation at 37 °C, fluorescence was measured on a multiwell plate reader (Varioskan LUX, Thermo Fisher). The experiment was repeated three times independently.

A minimum of 1 × 10⁵ cells were sorted out by flow cytometry based on the FITC-Annexin-V positivity. Then, following the manufacturer’s protocol, cells were harvested and measured with a Calpain Activity Fluorometric Assay Kit (Sigma Aldrich, MAK228-1KT).