Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells
Transkript
Bulk autophagy involves sequestering autophagic cargo and soluble cytosolic proteins, including lactate dehydrogenase or LDH, within autophagosomes for degradation.
To assess LDH sequestration, reflecting bulk autophagic activity, take an electroporation cuvette with nutrient-starved cells treated with inhibitors to block autophagic-lysosomal degradation.
Perform electroporation to selectively disrupt the cell membrane and release soluble proteins.
Mix the cell disruptate with buffered sucrose solution to preserve cellular components.
Transfer the required diluted disruptate solution to a buffer-containing tube. Centrifuge to separate non-sequestered LDH from autophagosome-sequestered LDH.
Transfer the remaining diluted disruptate solution, containing the total cellular LDH fraction, to another tube.
Add a non-ionic surfactant-containing buffer to both tubes to extract sequestered and total LDH fractions.
Centrifuge and transfer LDH-containing supernatants to buffer-containing tubes with pyruvate and NADH.
LDH converts pyruvate to lactate with NADH oxidation, decreasing NADH levels correlating with LDH concentration.
Quantify LDH in sedimented and total cellular fractions to determine LDH sequestration.
