For Leukemic CFC AssaysMethylcellulose-based Media
CFC Assay Media
The CFC assay has been used in the cancer stem cell field to study leukemic-CFCs and CFUs (for review see ref. 1). Like their normal counterparts, leukemic CFCs/CFUs require certain growth factors and nutrients supplied within a suitable three-dimensional matrix to grow distinct colonies. ColonyGEL™ is a family of high quality methylcellulose-based semi-solid media products for hematopoietic CFC assays. Different formulations are also suitable for growing various leukemic CFCs. While certain leukemic CFCs are morphologically distinct and can be visually distinguished from their normal counterparts, others look very similar to normal CFCs and can be confirmed as cancer CFCs by additional criteria in downstream assays (e.g. cancer cellspecific surface phenotype by flow cytometry; presence of leukemic genetic translocation by FISH analysis; serial replating ability in secondary CFC assays; etc.). This allows the evaluation of differential effects of anti-leukemia agents and other compounds on leukemic CFCs versus normal progenitors.
Examples of leukemic and normal CFC frequency in ColonyGEL™
CFU-GM and BFU-E from healthy and MM patient bone marrow (2x104 MNCs per dish) were grown in ColonyGEL™ 1102 (Human Complete Medium). For MM-CFCs, patient bone marrow MNCs were first immunomagnetically depleted of CD34+ and CD138+ cells (as per ref. 2) and plated at 2x105 cells per dish in ColonyGEL™ 1150 (Human Medium with PHA-LCM). This formulation is optimized for MM-CFC growth and contains a specially screened PHA-LCM as a source of growth factors. Cells from MM colonies were confirmed to express the MM surface marker CD138 by downstream flow cytometric analysis.
Acute Myelogenous Leukemia
CD34 analysis of normal and AML bone marrow MNCs was performed by flow cytometry. MNCs from normal and AML patient bone marrow (2x104 MNCs per dish) were cultured in ColonyGEL™ 1102 (Human Complete Medium). The AML CFUGM colonies grew as distinctive blast-like colonies (see picture on other side). BFU-E colonies were characteristically lacking in AML cultures.