I. Introduction:

Disruption of the mitochondrial transmembrane potential is one of the earliest
intracellular events that occur following induction of apoptosis. The MitoCaptureTM
Apoptosis Detection Kit provides a simple， fluorescent-based method for distinguishing
between healthy and apoptotic cells by detecting the changes in the mitochondrial
transmembrane potential. The kit utilizes MitoCaptureTM， a cationic dye that fluoresces
differently in healthy vs apoptotic cells. In healthy cells， MitoCapture accumulates and
aggregates in the mitocondria， giving off a bright red fluorescence. In apoptotic cells，
MitoCapture cannot aggregate in the mitochondria due to the altered mitochondrial
transmembrane potential， and thus it remains in the cytoplasm in its monomer form，
fluorescing green. The fluorescent signals can be easily detected by fluorescence
microscopy using a band-pass filter (detects FITC and rhodamine) or analyzed by flow
cytometry using FITC channel for green monomers (Ex/Em = 488/530+ 30 nm) and
(optional) PI channel for red aggregates (Em = 488/590+ 42 nm).

II. Kit Contents:

Component                                            K250-25                      K250-100
                                                               25 assays                  100 assays
MitoCapture Reagent                             25 μl                           100 μl
Incubation Buffer                                    50 ml                          2 x 100 ml

III. MitoCapture Assay Protocol:

A. General Considerations
Aliquot enough Incubation Buffer for the number of assays to be performed (total 2
ml for each assay) and pre-warm to 37oC before use.

B. Incubation of Cells with MitoCapture Reagent
1. Induce apoptosis in cells by desired method. Concurrently incubate a control
culture without induction.
2. Count cells and pellet ~1 x 106 cells per sample by centrifugation at 500 X g for 5
minutes.
3. Dilute MitoCapture Reagent immediately prior to use: Dilute 1 μl MitoCapture to 1
ml pre-warmed Incubation Buffer for each assay. Vortex the solution.
Note: MitoCapture is poorly soluble in aqueous solutions. To remove particles
(optional)， centrifuge the dye solution for 1 minute at 13，000 x g and carefully
transfer the supernatant without disturbing pelleted debris.
4. Resuspend cells in 1 ml of the diluted MitoCapture solution.
5. Incubate at 37oC in a 5% CO2 incubator for 15-20 min.
6. Centrifuge cells at 500 x g and discard supernatant.
7. Resuspend in 1 ml of the pre-warmed Incubation Buffer.

C. Quantification by Flow Cytometry
Analyze cells immediately following step B.7 by flow cytometry. MitoCapture
monomers in apoptotic cells are detectable in the FITC channel (usually FL1)
showing diffused green fluorescence. MitoCapture aggregates in healthy cells are
detectable in the PI channel (usually FL2) showing punctate red fluorescence.

D. Detection by Fluorescence Microscopy
1. Place the cell suspension from B.7 on a glass slide. Cover the cells with a glass
coverslip.
For analyzing adherent cells， grow cells on a coverslip and perform the entire
procedure directly on the coverslip in culture dish. Following incubation (B.7)，
invert coverslip on a glass slide.
2. Observe cells immediately under a fluorescence microscope using a band-pass
filter (detects fluorescein and rhodamine). MitoCapture that has aggregated in the
mitochondria of healthy cells fluoresces red. In apoptotic cells， MitoCapture cannot
accumulate in mitochondria， it remains as monomers in the cytoplasm， and
fluoresces green.

IV. Storage and Stability:
? Store MitoCapture at –20oC. Avoid freeze-thaw. Protect from light. Store
Incubation Buffer at 4oC after opening. All reagents are stable for 1 year.