Research Group of

Johannes Schmid

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Johannes A. Schmid

JC-1 stain of apoptotic cells

JC-1 (5, 5, 6, 6-tetrachloro-1, 1, 3, 3-tetraethylbenzimidazol-carbocyanine iodide) is a lipophilic fluorescent cation that incorporates into the mitochondrial membrane, where it can form aggregates due to the physiological membrane potential of mitochondria. This aggregation changes the fluorescence properties of JC-1 leading to a shift from green to orange fluorescence. Intact living cells stained with JC-1 therefore exhibit a pronounced orange fluorescence of mitochondria, which is detectable by flow analysis (in the FL-2 channel). Apoptosis results in a break-down of the mitochondrial membrane potential and a subsequent decrease of the orange fluorescence (and a slight increase of the green fluorescence). By that means, apoptotic cells can be easily distinguished from non-apoptotic cells.
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JC-1 is prepared as a 1000x stock solution in DMSO (5 mg/ml).
For the staining of adherent cells it is diluted in medium to 5 g/ml (with vortexing during the dilution to prevent the formation of precipitates); the JC-1 containing medium is added to the cells, followed by incubation for 10 min at 37C (or RT for 15 min).
Subsequently the cells are washed twice with PBS, trypsinized, suspended in 500 l PBS and analyzed by flow analysis.
Suspension cells (lymphocytes): suspend 1:1 with 10 g/ml JC-1 in medium (final conc.: 5 g/ml)

Approximate detection settings on FACSort:

FL1: 360 V (log)
FL2: 310 V  (log)
Compensation : FL1-7% FL2 und FL2-74% FL1

other settings: cell type specific, e.g. for TF-1:
SSC: 336 V lin
FSC: E00 lin 1.0
Threshold: FSC: 52 (or FL2)

Example of an analysis: