8% (v/v) formaldehyde directly into the well containing a
similar volume of DMEM medium.- The parameters, including PMT voltage, offset, pinhole,
and gain, should be kept unchanged between each picture
taken to ensure the accuracy of colocalization percentage
calculation. - Note that lysosomes or DRM fractions can also be used for
co-immunoprecipitation analysis (start fromstep 2) (Fig.1). - Make sure that the lysates are sufficiently sonicated; otherwise
protein aggregates will form during the immunoprecipitation. - The specific rounds of passing the lysates through the syringe
vary between different cell types. Six times of passing through
is sufficient for breaking MEFs. - Determine the efficiency of dephosphorylation at the end of
the reaction. - The cells should be rapidly rinsed. It is strongly recommended
to thoroughly rinse the cells: tilt the culture dish and apply the
mannitol solution (25 mL in total, per plate) to the upper side
of the dish by using a 25-mL serological pipette, and aspirate
the waste liquid at the bottom of the dish. - We find that it is better to label the dish on its bottom
rather than lid to prevent the lid from unscrewed in liquid
nitrogen. - The filter should be prevented from drying at all time.
- Avoid cervical dislocation. It has been reported to cause deg-
radation of ATP and ADP [33]. We anesthetize mice with
100 mg/kg sodium pentobarbital. Use of chloral hydrate is
also suitable for this assay. - Try to homogenate the tissue as quickly as possible to prevent
the degradation of ATP and ADP. - At the same time, cool the sample tray to 4C.
Acknowledgments
We thank the members of the Hai-Long Piao laboratory from
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
for the technical instruction on the CE-MS analysis of nucleotides.
This work was supported by grants from the National Key Research
and Development Project of China (2016YFA0502001) and
National Natural Science Foundation of China (#31430094,
#31690101, #31571214, and #31601152).Analysis of Lysosomal AMPK Activation 409