Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

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.

Using a pipette, resuspend the cell pellet to obtain a near-single cell suspension. Transfer this suspension to a 4-millimeter electroporation cuvette. Place the cuvette in an exponential decay wave electroporator and discharge a single electric pulse at 800 volts, 25 microfarads, and 400 ohms.

Use a new pipette tip to transfer the cell disrupting it into a 1.5-millimeter microcentrifuge tube containing 400 microliters of ice-cold phosphate-buffered sucrose solution, and mix briefly by pipetting. Repeat this resuspension, electrode disruption, and mixing process for each sample. If performing the procedure for the first time, make sure to verify efficient and selective plasma membrane electrode disruption as described in the text.

To obtain sedimented LDH fractions, remove 550 microliters from each diluted cell-disrupted solution, and transfer it to its own 2-milliliter microcentrifuge tube containing 900 microliters of ice-cold resuspension buffer with 0.5% BSA and 0.01% Tween-20. Pipette briefly to mix. Centrifuge at 18,000 times g and 4 degrees Celsius for 45 minutes to produce pellets containing sedimented LDH. Using suction, thoroughly aspirate the supernatant to leave the pellets as dry as possible.

Store the sedimented LDH samples at minus 80 degrees Celsius until ready to analyze. For total LDH fractions, transfer 150 microliters from each of the diluted cell-disrupted solutions to its own new tube. Store these in a minus 80 degrees Celsius freezer until ready to analyze.

Thaw both the sedimented LDH and the total LDH samples on ice. Once thawed, add 300 microliters of ice-cold resuspension buffer containing 1.5% Triton X-405 to the total LDH samples. Rotate the samples on a roller in a cold room for 30 minutes. Next, add 750 microliters of ice-cold resuspension buffer containing 1% Triton X-405 to the sedimented LDH samples. Use a pipette to resuspend the pellets until the solution is homogeneous. Centrifuge all of the samples at 18,000 times g in 4 degrees Celsius for five minutes to sediment undissolved cellular debris.

After centrifugation is complete, put the samples on ice. Determine the levels of LDH in the supernatants using either a classic enzymatic assay as described in the text or any of the wide variety of commercially available kits.