Quantifying Apoptosis in Primary AML Samples

Annexin V and PI (purchased from Sigma-Aldrich)-binding assays were performed to assess apoptosis as described previously [40 (link)]. Annexin V-negative and PI-negative cells were counted as live cells. Apoptosis (A) in a sample was quantified as the proportion of cells that were Annexin V-positive cells, and specific apoptosis was calculated by the following formula: %specific apoptosis = (A^test − A^control) × 100/(100 − A^control). Thus, by subtracting spontaneous apoptosis (= apoptosis observed in untreated control samples), “specific apoptosis” in the present study indicates specifically induced apoptosis by RHT. For gating stem/progenitor population (CD45+CD34+CD38− cells) in primary AML samples, phycoerythrin (PE)-conjugated anti-human CD45, PE-Cyanine7 (PE-Cy7)-conjugated anti-human CD34, and fluorescein isothiocyanate-conjugated CD38 antibodies and Allophycocyanin-conjugated Annexin V (BD Biosciences, San Jose, CA) were used.