Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells
Trascrizione
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.
