Xmark

Cell viability was measured according to mitochondrial dehydrogenase activity tested by standard procedure based on the reduction of MTT tetrazolium dye. Cells (10⁴ per well in 96-well plate, CELLTREAT Scientific Products, United States) have grown overnight, then cultural fluid was discarded and fresh medium with test samples was added. After 24 h, formazan absorption was measured (xMark, Bio-Rad, United States).

Activation of caspase-3 was measured using a colorimetric caspase-3 assay kit (CaspACE^TM Assay System, Promega, Madison, WI, USA). Briefly, exponentially growing HL-60 cells were treated with tested derivatives (10–30 µM) for 48 h. Cells (5 × 10⁶) were pelleted by centrifugation (1200× g, 3 min at 4 °C) and resuspended in 50 μL of lysis buffer (Promega, Madison, WI, USA). The lysed cell mixture was then incubated on ice for 15 min before centrifugation (13,000× g, 5 min at 4 °C). 42 μL of reaction buffer supplemented with 10 mM DTT were mixed with 20 µL of cell lysates. The substrate DEVD-pNA (2 µL) was added and the samples were incubated for 4 h at 37 °C. The release of p-nitroaniline from specific caspase-3 substrate was measured at 405 nm using a microplate reader (xMark, BioRad, Tokyo, Japan).

The relative cell viability was determined by the Cell Counting Kit-8 (CCK-8 Kit, Dojindo, Dalian, China) according to the provider’s manual. 10,000 cells were plated into each well of 96-well plate in triplicate for 72 h. 10 µL of CCK-8 solution was subsequently applied into each well and allowed for reaction at 37 °C for 1 h. The absorption at 450 nm was recorded by microplate reader (Xmark, Bio-Rad, CA, USA) and relative cell count was calculated.

Total peptide concentrations in anionic and cationic compartments were determined using micro BCA (µBCA) protein assay (Pierce, Rockford, IL, USA) from the samples withdrawn every 30 min over a period of 120 min. The absorbance was read at 562 nm on a microplate reader (xMark, Bio-Rad, Hercules, CA, USA). Concentration was determined with a standard curve in a range of 0-40 μg/mL of bovine serum albumin (BSA) [15 (link)].
For all FM, global peptide migration rate (g/m²·h) was calculated by dividing the total amount of peptides (g) migrated to anionic recovery compartment (A⁻_RC)/cationic recovery compartment (C⁺_RC) at the end of EDFM, determined by µBCA, by effective surface area of FM (m²) and duration of EDFM process (h). High concentration of peptides in recovery compartments is associated with its high migration rate in that compartment.

Glucose levels in plasma were measured with colorimetric assay (Dojindo, Kumamoto, Japan). All samples were assayed in duplicate. Optical density at 450 nm was measured using an xMark microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). The sample concentrations were estimated using a linear fit of the log–log plot of the standard curve.