JC-1

Protocol for mitochondrial membrane potential cell assay ^[1]:

1. Prepare a 5mg/mL stock solution of JC-1 in DMSO (~7.7 mM). JC-1 is light sensitive. Do not expose to direct intense light. Perform all staining procedures in subdued light. Avoid repeated freeze/thawing of the JC-1 stock solution. Make small aliquots after the first thaw and store them at-20℃.
2.  Grow cells on a glass cover slip in a petri dish or in a chamber slide. Induce the cells according to your specific protocol.
3. Warm fresh culture medium to 37℃. Thaw an aliquot of the JC-1 stock solution at room temperature (RT). Make sure JC-1 is completely thawed and warmed to RT before diluting. Mix thawed JC-1 well before dilution. Prepare the JC-1 staining solution by diluting the reagent in pre-warmed culture media. For the staining of adherent cells, JC-1 is diluted in medium to a final concentration of ~ 5 ug/mL (vortex during dilution to prevent the formation of precipitates). Mix well to dissolve all particulates. Do not centrifuge the reagent. Dilute JC-1 reagent immediately prior to use.
4. Remove the cell culture media and replace with enough diluted 1X JC-1 reagent sufficient to cover the cells.
5. Incubate the cells for 10 min at 37℃ (or RT for 15 min). The duration of the staining depends upon the cell type.
6. Remove the media and wash the monolayer twice with PBS or fresh media.
7. Add a drop of PBS and cover the cells with a cover slip.
8. Observe cells immediately with a fluorescence microscope using a dual-band pass filter designed to simultaneously detect fluorescein and rhodamine or fluorescein and Texas Red. In live non-apoptotic cells, thel mitochondria will appear red following aggregation of the JC-1 reagent. The red aggregates emit at about 590 nm. In apoptotic and dead cells the dye will remain in its monomeric form and will appear green with an emission at about 530 nm.

1. 在DMSO (~7.7 mM)中配制5mg/mL的JC-1原液。JC-1是光敏的。不要直接暴露在强光下。在弱光下进行染色。避免重复冻结/解冻JC-1原液。在第一次解冻后制作小份，并在20°C下保存。

2. 在培养皿或室内载玻片的玻璃罩上培养细胞。按照方案诱导细胞。

3.将新鲜培养基加热至37°C。在室温(RT)下解冻一份JC-1原液。在稀释之前，确保JC-1完全解冻并加热到RT。在稀释之前充分混合解冻的JC-1。在预热的培养基中稀释试剂，制备JC-1染色液。为了对贴壁细胞进行染色，将JC-1在培养基中稀释至终浓度为~ 5 ug/mL(稀释时涡旋，以防止沉淀形成)。充分混合，不要离心。使用前立即稀释JC-1试剂。

4. 取出细胞培养基，替换为足够稀释的1x JC-1试剂，覆盖细胞。

5. 37℃孵育细胞10分钟(或RT孵育15分钟)。染色的持续时间取决于细胞类型。

6. 除去培养基，用PBS或新鲜培养基清洗细胞两次。

7. 加入一滴PBS，用盖片覆盖细胞。

8. 立即用荧光显微镜观察细胞，使用设计用于同时检测荧光素和罗丹明或荧光素和德州红的双带通滤镜。在活的非凋亡细胞中，随着JC-1试剂的聚集，线粒体将呈现红色。红色聚集体的发射波长约为590nm。在凋亡细胞和死亡细胞中，染料将保持其单体形式，并在约530nm处呈现绿色。

Protocol for functional assay JC-1 for P-glycoprotein (Pgp) ^[2]:

1. For staining, cells were washed twice and resuspended in PBS containing 0.1 μM JC-1 monomer at a concentration of 5x10⁵ cells/mL and incubated at 37℃ for 15 without or with modulator to assess Pgp function.
2. Cells were washed twice in cold PBS, and samples were analyzed.
3. Cell fluorescence was recorded using a FACSort flow cytometer equipped with a 488-nm argon laser and 3 fluorescence detectors: FL1 (530-nm band-pass filter), FL2 (585-nm band-pass filter), and FL3 (650 nm band-pass filter).
4. JC-1 fluorescences were analyzed on the FL1 and FL2 channels for detection of the fluorescence of the dye monomer and liquid crystal form, respectively. The function of Pgp was established with blast cells selected by CD34 antibody or with physical characteristics only if blast cells did not express characteristic markers.

1.    染色时，将细胞洗涤2次，重悬于含有0.1 μM JC-1单体的PBS中，浓度为5 × 10⁵个/mL, 37℃孵育15天，不加或加调制剂，以评估Pgp的功能。

2.    细胞在冷PBS中洗涤两次，并对样品进行分析。

3.    使用FACSort流式细胞仪记录细胞荧光，该流式细胞仪配备488 nm氩激光器和3个荧光检测器:FL1 (530 nm带通滤波器)、FL2 (585 nm带通滤波器)和FL3 (650 nm带通滤波器)。

4.    分析JC-1在FL1和FL2通道上的荧光，分别检测染料单体和液晶形态的荧光。通过CD34抗体选择的母细胞或不表达特征标记物的母细胞建立Pgp功能。

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1].  Perelman A, Wachtel C, et,al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 2012 Nov 22;3(11):e430. doi: 10.1038/cddis.2012.171. PMID: 23171850; PMCID: PMC3542606.

[2].  Legrand O, Perrot JY, et,al.  JC-1: a very sensitive fluorescent probe to test Pgp activity in adult acute myeloid leukemia. Blood. 2001 Jan 15;97(2):502-8. doi: 10.1182/blood.v97.2.502. PMID: 11154229.