KMG-301AM

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs)^[1].
1. For optical imaging, 20 μM KMG-301AM is applied to cells in Hanks' balanced salt solutions (HBSS) for 10 min on ice, so that hydrolysis of the acetoxymethyl ester by esterase present in the cytosol will be avoided.
HBSS contains (in mM): NaCl, 137; KCl, 5.4; CaCl₂, 1.3; MgCl₂, 0.5; MgSO₄, 0.4; Na₂HPO₄, 0.3; KH₂PO₄, 0.4; NaHCO₃, 4.2; D-glucose, 5.6; HEPES, 5 (pH adjusted to 7.4 with NaOH).
2. Then, the cells are washed twice with HBSS and incubated for 15 min at 37°C to allow for complete hydrolysis of the acetoxymethyl ester form in mitochondria.
3. If you want the simultaneous use of KMG-104AM (HY-128536) and KMG-301AM, cells are incubated with 5 μM KMG-104AM in HBSS for 30 min at 37°C, and then stained with KMG-301AM.
4. Isolated mitochondria are incubated with 20 μM KMG-301AM in Mitochondria Imaging Buffer (MIB) for 20 min at 37°C. Then, mitochondria are washed twice with MIB and further incubated for 15 min at 37°C.
MIB contains (in mM): KCl, 125; K₂HPO₄, 2; MgCl₂, 1; HEPES, 5; EDTA, 0.02 (pH adjusted to 7.2 with KOH) or Mg²⁺-free MIB (without MgCl₂)
5. Fluorescence imaging experiments are performed using a confocal laser scanning microscope system. KMG-301 is excited at 559 nm from a laser diode, and the signal was observed at 600-700 nm.
In the simultaneous measurement, KMG-104 and KMG-301 were excited at 488 nm from an Argon laser and 559 nm, respectively. Fluorescence was separated using a 560 nm dichroic mirror and observed at 500-545 nm and 600-700 nm, respectively.

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