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PROTOCOLS
Analysis of mitochondrial membrane potential
Accuri Cytometers
Sponsored,vendor-submitted protocol    Sponsored by Accuri Cytometers    Published in November 2009 2009 (p.17) DOI: 10.2144/000113278

Abstract

Flow cytometry is the technique of choice for analysis of mitochondrial transmembrane potential (ΔΨm) in whole cells. JC-1, a cytofluorimetric, lipophilic cationic dye, selectively enters into mitochondria and reversibly changes color from red to green as the membrane potential decreases. In healthy cells with high ΔΨm, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. In apoptotic or unhealthy cells with low ΔΨm, JC-1 remains in the monomeric form, which shows only green fluorescence.

Sample setup

It is possible to co-label cells with JC-1 and antibodies for cell surface markers or other viability markers, as long as the fluorescence emission of the additional label is spectrally resolved from JC-1. JC-1 fluorescence is measured in the FL1 (530 +15 nm) and FL2 (585 +20 nm) channels of the Accuri C6 Flow Cytometer®.

Cells from each experimental group should be treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to create a strong, single-positive green fluorescence control. CCCP causes quick mitochondrial membrane depolarization, greatly reducing the FL2 red fluorescence signal, thus providing a green signal–only positive control. An unstained control sample for each cell type and experimental condition should be included.

Materials and methods

JC-1 Mitochondrial Membrane Assay Kit (PN KR310; Accuri Cytometers)

Cytometric analysis

Figure 1, Table 1. Sample set-up. (A) Light scatter gate and (B) FL1 vs. FL2 for cells with JC-1 and without CCCP (Tube 2). (C) Zoom of FL1 vs. FL2 with P2 gate drawn.

1. Set FSC-H threshold to channel 80,000.

2. Create a FSC vs. SSC density plot.

a. For smaller cells use linear FSC (channel range 0–1.6 × 106) vs. linear SSC (channel range 0–800,000) as a starting point.

b. For larger cells, use log FSC vs. log SSC, and then zoom in around the main cell population after data collection.

3. Create a log FL1 (x-axis) vs. log FL2 (y-axis) density plot.

4. Collect 50,000 –100,000 total events from the controls and samples.

5. View data for Tube 1, the unstained control (Table 1).

6. Draw a polygon gate (P1) around the cells, excluding debris, on the FSC vs. SSC plot.

7. Apply “P1 in all events” to the FL1 vs. FL2 plot.

8. View staining of cells with JC-1 using Tube 2 (Table 1). Adjust P1 if needed (Figure 1).

9. The FL1 vs. FL2 plot should now contain a large, double-positive population.

10. Adjust the FL1 axis view with Zoom or Plot Spec Tools. Draw a polygonal gate, (P2) around the main population.

11. View JC-1 staining of Tube 3. The percentage of cells in the P2 gate for the CCCP control will be the “background” value to be subtracted from the percentage of P2 events in all test samples of the applicable treatment group.

12. If an unacceptable percentage of cells from the CCCP control falls inside P2, using the CFlow® Zoom Tool to precisely adjust the P2 gate to exclude background is effective without the need for fluorescence compensation.

a. Zoom in to magnify the region of the JC-1-FL1 vs. JC-1-FL2 plot where the FL2- and FL2+ populations seem to overlap.

b. Adjust the perimeter of P2 to more precisely exclude background from the CCCP control sample. Zoom out.

13. While viewing the data for the CCCP control, draw a polygon gate (P3) around the major population of cells, and then adjust the lower edge of P2 to meet (but not overlap) the upper edge of P3.

14. Set an appropriate run limit in CFlow (from 10,000 to 50,000 cells in P1) and collect the data for all remaining samples.

15. The appropriate P1, P2, and P3 regions within each experimental group may need to be adjusted, as light scatter and JC-1 staining characteristics may change with treatment.

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