JC-1 - Mitochondrial Membrane Potential Assay Kit (ab288313)

JC-1 assay result in HL60 cells treated with FCCP.  HL60 cells were seeded and labeled according to section 11.1 of the protocol.  Cells were then treated for 4 hours with 100 µM FCCP or vehicle/diluent control (DMSO).  Cells were read on a Perkin Elmer-Wallac 1420 Victor 2 Multilabel plate reader. Mean and standard deviation is plotted for 3 replicates from each condition. 

JC-1 assay result in HepG2 cells treated with CCCP. HepG2 cells were seeded and labeled according to section 11.2 of the protocol.  Cells were then treated for 4 hours with a titration series of CCCP (carbonyl cyanide 3-chlorophenylhydrazone) and both monomer and aggregate forms were read on a Perkin Elmer-Wallac 1420 Victor 2 Multilabel plate reader.  Mean and standard deviation of aggregate/monomer ratios is plotted for 12 replicates for each concentration.  IC₅₀ of CCCP in HepG2 cells was calculated at 8.7 µM

JC-1 assay result in HL60 cells treated with Troglitazone. HL60 cells were seeded and labeled according to section 11.1 of the protocol.  Cells were then treated for 4 hours with a titration series of the thiazolidinedione Troglitazone and both monomer and aggregate forms were read on a Perkin Elmer-Wallac 1420 Victor 2 Multilabel plate reader.  Mean and standard deviation of aggregate/monomer ratios is plotted for 3 replicates for each concentration.  IC₅₀ of Troglitazone in HL60 cells was calculated at 1.2 µM

HeLa cells plated at 12000 cells/well. Next day, aspirated media and replaced with complete media containing 0, 25, 50, or 100uM FCCP for 30 minutes. Drug solution aspirated from wells and cells were washed once with serum free media. A staining solution of 1uM  JC-1 in serum free media was added to cells and incubated for 15 minutes at 37°C in the dark. Staining solution was aspirated, cells were washed once with 1X PBS, and bathed in 1X Opti-klear. Measured fluorescence on Tecan plate reader using Ex. 485/Em. 530 for monomer and Ex. 535/Em. 590 for aggregates.

HeLa cells plated at 12000 cells/well. Next day, aspirated media and replaced with complete media containing 0, 25, 50, or 100uM FCCP for 30 minutes. Drug solution aspirated from wells and cells were washed once with serum free media. A staining solution of 1uM JC-1 in serum free media was added to cells and incubated for 15 minutes at 37°C in the dark. Staining solution was aspirated, cells were washed once with 1X PBS, and bathed in 1X Opti-Klear. Plate was analyzed using the CX5 CellInsight SpotDetector bioapp

GM14643 healthy leukocytes were treated with 0, 25, 50, or 100uM FCCP in complete media for 30 minutes. Cells were collected by centrifugation and washed once with serum free media. Cells were resuspended with a staining solution of 5uM or 10uM JC-1 in serum free media and incubated for 30 minutes at 37°C in the dark. Cells were collected by centrifugation, washed once with 1XPBS, and resuspended in 1X Opti-Klear. Cells were then seeded in a 96-well optical bottom plate at a density of 200,000 cells/50uL/well. Fluorescence was measured on Tecan plate reader using Ex. 485/Em. 530 for monomer and Ex. 535/Em. 590 for aggregates.

GM14643 healthy leukocytes were treated with 0, 25, 50, or 100uM FCCP in complete media for 30 minutes. Cells were collected by centrifugation and washed once with serum free media. Cells were resuspended with a staining solution of 5uM or 10uM JC-1 in serum free media and incubated for 30 minutes at 37°C in the dark. Cells were collected by centrifugation, washed once with 1XPBS, and resuspended in 1X Opti-Klear. Cells were then seeded in a 96-well optical bottom plate at a density of 200,000 cells/50uL/well. Plate was analyzed using the CX5 CellInsight SpotDetector bioapp

HeLa cells plated at 12000 cells/well. Next day, aspirated media and replaced with complete media containing 0 or 100uM FCCP for 30 minutes. Drug solution aspirated from wells and cells were washed once with serum free media. A staining solution of 1uM JC-1 in serum free media was added to cells and incubated for 15 minutes at 37°C in the dark. Staining solution was aspirated, cells were washed once with 1X PBS, and bathed in 1X Opti-Klear. Plate was imaged using the CX5 CellInsight.

GM14643 healthy leukocytes were treated with 0, 25, 50, or 100uM FCCP in complete media for 30 minutes. Cells were collected by centrifugation and washed once with serum free media. Cells were resuspended with a staining solution of 5uM or 10uM JC-1 in serum free media and incubated for 30 minutes at 37°C in the dark. Cells were collected by centrifugation, washed once with 1XPBS, and resuspended in 1X Opti-Klear. Cells were then seeded in a 96-well optical bottom plate at a density of 200,000 cells/50uL/well. Plate was imaged using the CX5 CellInsight.