Cytotox glo cytotoxicity assay kit

NHL cells were incubated with 10 μg/mL anti-CD20 mAbs or PPRT nanocombs. For CDC assays, 5% (v/v) fresh human serum (donated by Dr Zheng Xu) was employed as a source of complement, while, for ADCC assays, human peripheral blood mononuclear cells (donated by Dr Cong Wu) were added as effector cells with an effector/target ratio of 25:1. After a 4-hour incubation, dead cells were assessed using CytoTox-Glo™ Cytotoxicity Assay kit (Promega Corporation) following the product information. Cells lysed by the lysis reagent (digitonin) offered in the assay kit were used as positive controls.

Cytotoxicity of T cells induced by BiTE was measured using the dead-cell protease release method. Briefly, target cells (10,000 cells/well) were mixed with serially diluted BiTE antibodies in 96-well white plates (Corning) in 50 μL of growth medium. Activated T cells (50 μL) were added to the wells at effector to target (E:T) ratio 10:1 and cultured for 24 h at 37 °C in a 5% CO₂ humidified atmosphere. CytoTox-Glo™ Cytotoxicity Assay kit (Promega, Madison, WI) was used to measure the relative number of dead cells in cell populations following the manufacturer's instructions. A maximum lysis control was used where digitonin was added to target cell only wells before adding the lysis reagent. The luminescence signal was measured using BioTek synergy multi-mode reader (Winooski, VT). The percent cytotoxicity was calculated using the following formula:% Lysis = 100 × (Experimental signal – Untreated cells signal)/(Maximum signal – Untreated cells signal). For CHO-K1-CD276-Luci cells, the luciferase signal was measured using Bright-Glo Luciferase Assay System (Promega, Madison, WI) following the manufacturer's instructions. The percent cytotoxicity was calculated using the following formula:% Lysis = 100 × (1-Experimental signal / Untreated cells signal). Curves were fitted using a four-parameter logistic fitting with GraphPad Prism 8.

A BrdU incorporation assay was performed to analyze the cell proliferation (cycle) of UT7/Epo-S1 cells and CD36⁺ EPCs, followed by flow cytometry analysis, as described previously (57 (link)). The cell viability assay was performed as previously described using a CytoTox-Glo cytotoxicity assay kit (#G9290, Promega) (58 (link)).