JC-1 (CBIC2) is a fluorescent lipophilic carbocyanine dye used to measure mitochondrial membrane potential. JC-1 forms complexes known as J-aggregates at high ΔΨm. Aggregates of JC-1 emit an orange-red fluorescence (Ex/Em=585/590 nm). While in cells with low ΔΨm, JC-1 remains in the monomeric form. JC-1 monomers emit a green fluorescence (Ex/Em=510/527 nm).

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Labeling of Cells:
1. Culture cells in 6-, 12- , 24-, or 96-well plates at a density of 5× 10⁵ cells/mL. Incubate the cells according to your normal protocol.
2. Ensure that the JC-1 and DMSO has equilibrated to room temperature, and then prepare a 200 μM stock solution by dissolving the contents of one vial in DMSO provided.
3. For the control tube, allow the vial of CCCP has come to room temperature, add 1 μL of CCCP (50 mM). Incubate cells at 37°C for 5 minutes.
4. Add 10 μL JC-1 (200 μM) per well to make the final concentration at 2 μM. Incubate cells at 37°C, 5% CO₂, for 15-20 minutes. If additional labeling followed, for example with an annexin V, begin with step 2.a. If not, proceed with step 1.e.
5. After incubation, centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant.
6. Wash cells twice with PBS (1×): add 2 mL PBS (1×) to suspend cells and vortex to mix thoroughly. Centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant.
7. Add 500 μL PBS (1×) to suspend cells. Analyze sample on a flow cytometer, fluorescence microscopy, or fluorescence microplate reader.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

652.23

C₂₅H₂₇Cl₄IN₄

3520-43-2

Room temperature in continental US; may vary elsewhere.

4deg;C, sealed storage, away from moisture and light

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (sealed storage, away from moisture and light)

In Vitro:;

DMSO : 5 mg/mL (7.67 mM; ultrasonic and warming and heat to 60°C)

H₂O : < 0.1 mg/mL (insoluble)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂：;10% DMSO ;; 40% PEG300 ;; 5% Tween-80 ;; 45% saline

  Solubility: 1.25 mg/mL (1.92 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.25 mg/mL (1.92 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 12.5 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂：;10% DMSO ;; 90% (20% SBE-β-CD in saline)

  Solubility: 1.25 mg/mL (1.92 mM); Suspended solution; Need ultrasonic

  此方案可获得 1.25 mg/mL (1.92 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  以 1 mL 工作液为例，取 100 μL 12.5 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. A Perelman, et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 2012 Nov 22;3:e430.

  [2]. Vera C. Keil, et al. Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria. Pflügers Archiv – European Journal of Physiology. 2011,462(5): 693-708.

  [3]. Jung-Ho LEE, In-Hwan LEE, Young-Jun CHOE, et al. Real-time analysis of amyloid fibril formation of α-synuclein using a fibrillation-state-specific fluorescent probe of JC-1. Biochem. J. 2009, 418:311-323.

  [4]. Salvioli S, et al. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1997 Jul 7;411(1):77-82.