Cell‐mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere‐adjusted counts on three‐color flow cytometry

Abstract

Background: In addition to 51 chromium release assay, flow cytometric methods have been described to assess in vitro cell-mediated cytotoxicity. In this report, we describe a new flow cytometric approach for determination of in vitro cell-mediated cytotoxicity utilizing three-color flow cytometric assay. Methods: This method is based on monoclonal antibody staining of either effector or target cells to evaluate cytotoxicity with increased accuracy by utilizing fluorospheres for calibration. The basic strategy involves labeling effector or target cells with a specific fluorescentconjugated monoclonal antibody, in addition to staining with annexinV-FITC and propidium iodide to identify apoptotic/dead cells. The effector and target cell populations as well as conjugates were clearly and easily identified by this approach. Results: We obtained significant correlation between cytotoxicity calculated by this technique and 51 chromium

Rebuilding Human Leukocyte Antigen Class II–Restricted Minor Histocompatibility Antigen Specificity in Recall Antigen-Specific T Cells by Adoptive T Cell Receptor Transfer: Implications for Adoptive Immunotherapy

“…To determine the lytic activity of T-cell clones on B cells and on MM cells at long term, we slightly modified a previously described FACS-based cytotoxicity assay (19). T-cell clones and target cells (EBV-LCLs or MM cells) were coincubated at different ratios in a final volume of 200 AL RPMI/10% FBS at 37jC.…”

Irradiated KHYG-1 retains cytotoxicity: Potential for adoptive immunotherapy with a natural killer cell line

“…A total of 10 4 cells/ reaction were analysed for each sample at each time point. Percent lysis was reverse calculated from the viable cells at a given time point compared to time 0 h, excluding 7AAD and Annexin V-FITC positive cells, according to the formula [time 0 h (mean) -time point h (mean)/time 0 (mean) 6 100] (Ozdemir et al 2003) and the standard error of the mean (SEM) was calculated. Significance for the results is expressed as p-values determined by Student's t-test for pairwise comparisons.…”

Identification of Mixed Lineage Leukemia 1(MLL1) Protein as a Coactivator of Heat Shock Factor 1(HSF1) Protein in Response to Heat Shock Protein 90 (HSP90) Inhibition

“…4, H and I) did not sensitize cells to those two inhibitors, suggesting that Mll1/Men1 knockout has selective effects on sensitizing to heat shock pathway induction. To further understand the mechanism of the combination effect between MLL1 knockout and HSP90 inhibition, we assayed for cell death and apoptosis by FACS subsequent to staining cells with 7AAD (a marker for dead cells) and Annexin V (a marker for cell apoptosis) (38). This analysis showed that more than 80% of Mll1 Ϫ/Ϫ MEFs were apoptotic compared with only 30% of Mll1 ϩ/ϩ cells after exposure to AUY922 treatment (Fig.…”