Protocol for separating cancer cell subpopulations by metabolic activity using flow cytometry

Abstract

Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations. For complete details on the use and execution of this protocol, please refer to Blaszczak et al.¹.

Keywords: Cancer; Flow Cytometry; Metabolism.

Graphical abstract

Figure 1

Schematic of protocol to calibrate HPTS and RuBPY fluorescence

Figure 2

Gating strategy Step 1: Draw a gate around the bulk of the cell population. Step 2: Exclude DAPI-stained (i.e., dead) cells to ensure that only living cells are analyzed and collected. Step 3: Exclude cell doublets. Step 4: Draw a gate that represents alkaline cells (i.e., cells of high lactate efflux capacity, corresponding to higher metabolic rate) and a gate around acidic cells (i.e., cells of low lactate efflux capacity, corresponding to lower metabolic rate).

Figure 3

Exemplary data depicting metabolic heterogeneity of the MIA PaCa-2 cell line Panel A shows a series of scatter plots of pH in cells during evoked lactate efflux. The alkaline population (red; A) corresponds to the sub-population of cells with higher lactate efflux capacity, while the acidic sub-population (blue; B) describes cells with lower lactate efflux capacity. Note, the transient character of the alkaline sub-population. Panel B depicts the distinct metabolic phenotypes of collected subpopulations in terms of medium pH and medium oxygen, cumulative acid production and oxygen consumption. For clarity, a single representative repeat is shown.

Figure 4

The pH-sensitivity of buffering capacity of the low buffering medium