Apoptosis Detection Procedure III--JC-1 Detection of Mitochondrial Membrane Potential Changes in Apoptotic Cells

May 15, 2023

Apoptosis detection operation flow
3. JC-1 detects changes in mitochondrial membrane potential of apoptotic cells

BDTM MitoScreen (JC-1) Mitochondrial Membrane Potential Detection Kit (551302):

Component description
JC-1 (dye) 4 bottles, each for 25 samples. Freeze-dried powder, diluted to stock solution before use (Stock Solution), diluted to working solution (Working Solution)
10 x Assay Buffer 60ml, enough for 100 samples. Dilute to 1X before use

Reagent preparation:  

A.10× Assay Buffer dilution (Assay Buffer as the experimental reaction solution for washing cells):  

a) Place 10× Assay Buffer in a 37 ° C water bath until dissolved.
b) Dilute 10× Assay Buffer 1:10 with double distilled water and stir for 5 minutes.
c) Preheat the 1× Assay Buffer at 37 °C before use.
Preparation of JC-1 stock solution (one bottle):
a) Dilute JC-1 lyophilized powder in 125 μl DMSO per bottle at room temperature. The inverted vial was repeatedly inverted and the JC-1 was fully dissolved.
b) The stock solution should be diluted into working fluid immediately or dispensed into small portions (protected from light) and stored at -20 °C (up to 6 months).
c) Avoid repeated freezing and thawing of JC-1 stock solution.

Preparation of JC-1 working fluid:
a) Preheat 1 x Assay Buffer at 37 °C.
b) Dilute the JC-1 stock solution to JC-1 working solution with 1× Assay Buffer 1:100. For example, 125 μl of JC-1 stock solution is added to 12.375 ml of preheated 1× Assay Buffer. For the experimental procedure, 0.5 ml of JC-1 working solution was required for each sample (<106 cells).
c) Gently mix the JC-1 working solution for use. (A small amount of JC-1 aggregated particles may appear after mixing, but it does not affect flow detection. It can also be removed by centrifugation at 13,000 g for 1 minute at room temperature or centrifugation at 1,000 g for 15 minutes for removal of JC-1.

Experimental operation:

1. Prepare a cell suspension of 1x106 /ml, the concentration should not be too high, because it is easy to cause apoptosis of cells beyond this concentration.

2. Inducing cells to undergo apoptosis treatment while preserving an uninduced cell as a control.

3. After the apoptotic process, each 15ml sterile polystyrene centrifuge tube 1ml cell suspension was added, at room temperature, 400 × g, 5 minutes centrifugation, the supernatant was discarded.

4. Add 0.5 ml of freshly prepared JC-1 working solution to each tube, mix well, place in a 37 ° C CO2 incubator, and incubate for 10-15 minutes.

5. Wash the cells twice as follows:
  First: added to each tube a volume of 2 ml of 1 × Assay Buffer, gently resuspend the cells with a pipette or shaken to disperse the cells, the cells in order to avoid poly manifold. 400 x g, centrifuge at room temperature for 5 minutes, discard the supernatant.
Second: 1ml added to each tube in a volume of 1 × Assay Buffer, gently resuspend the cells with a pipette or shaken to disperse the cells, the cells in order to avoid poly manifold. 400 x g, centrifuge at room temperature for 5 minutes, discard the supernatant.

6. Add 0.5 ml of 1× Assay Buffer to each tube, gently suspend the cells and test on the machine.

 

For more products you can contact us by:

Free hotline: 4008-168-068 phone / 89
Email:       Website:
Public number: You Ning antibody expert

Extract Powder

China Extract Powder For Use As Dietary Supplement Extract Powder, Extract Powder Manufacturer

Shaanxi Kang New Pharmaceutical co., Ltd. , https://www.kangnewpharmas.com