Wst 1 assay kit

Cell proliferation was measured using a WST-1 assay kit (BioVision, Milpitas, CA, USA). The cells were cultured in 96-well culture plates (8 × 10³ cells/well). WST-1 reagent was added to the wells of the experimental and control groups at 6, 12, 24, and 48 h. After incubation for 3 h (37 °C, 5% CO₂), an aliquot of culture supernatant was measured at 450 nm with a Multiskan™ FC Microplate Photometer (Molecular Devices, Sunnyvale, CA, USA). The absorbance of WST-1 reagent without cells was used as the blank.

The cell proliferation assay was performed using a WST-1 Assay Kit (BioVision, Inc., Milpitas, CA, USA). A total of 400 HNE-1 and CNE2 cells were plated onto 6-well plates and incubated at 37 °C in a 5% CO₂ incubator for 2 weeks. Fresh medium was added every 3 days. At the end-point, the cells were washed twice with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 minutes, and stained with 1% crystal violet solution for 20 minutes at room temperature. The visible colony numbers were counted. This experiment was performed in triplicate.

Cytotoxicity against human leukemia (CCRF-CEM), acute promyelocytic leukemia (NB4), acute monocytic leukemia (THP1), and human lymphoma (U937) was determined using the Abcam^® Water Soluble Tetrazolium Salts (WST-1) assay kit (ab155902 WST-1 Cell Proliferation Reagent, UK). Cell suspensions were incubated for 24 h, and then treated with different concentrations (0 to 100 μg/mL in complete media) of mushroom extracts for 48 h. Cells were then treated with 10 μL WST-1 reagent for 1 h, then A₄₅₀ was read using a microplate reader [38 (link)].

Cell proliferation was measured by the WST-1 assay kit (ab155902; Abcam, Cambridge, MA, USA) or CyQUANT cell proliferation assay kit (Invitrogen, Carlsbad, CA, USA). Briefly, three thousand cells were seeded into each well of a 96-well plate in RPMI 1640 medium with 10% FCS for 24 h. The culture media were changed to serum-free medium with/without 10 ng/mL TGFβ or 20 μM CAPE for 24 h. For WST-1 cell proliferation cells, a 10 μL of WST-1 reagent was added directly into the culture medium and cells were incubated for 1 h before the amount of formazan dye was produced by measuring the absorbance at 440 nm using the synergy H1 microplate reader (BioTek Instruments, Inc., Beijing, China). For the CyQUANT cell proliferation assay, after cells were washed twice with phosphate-buffered saline (PBS), the cell pellet was frozen at −80 °C for 1 h. Then, 200 μL of CyQUANT GR dye with lysis buffer (Invitrogen) was added to each well and incubated for 10 min at room temperature before the fluorescence was measured at 488 nm excitation using the synergy H1 microplate reader (BioTek Instruments Inc., Beijing, China).

The potential cytotoxic induction of both SOR and H-P extract in treated cells was achieved via monitoring the cell morphology and accounting the number of survived cells upon treatment. The 50% cytotoxic concentration (CC₅₀) of each effector and cell viability rate of treated cells were assessed using WST-1 assay kit (Abcam, USA). Briefly, the same volume of WST-1 reagent was added to 100 µl fluid media of the treated cells in the enzyme-linked immunosorbent assay (ELISA) reader plate and was incubated for 2 h away from the light, and then, the formazan product was measured at the wavelength of 440 nm using an ELISA plate reader (IndiaMART, Delhi, India). Assessing released LDH within the fluid medium of the transfected cells was monitored in a 96-well plate using the LDH production kit (Abcam, ab102526). Following the manufacturing procedures, 100 µl of each sample was incubated with 40 µl LDH buffer and 20 µl substrate for 1 h. Then, the relative production of LDH was calculated by dividing the mean absorbance values of treated cells by the mean absorbance values of the mock, which was indicated by the fold change (23 (link)).