Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

1. Cell Seeding and Treatment

1. Culture adherent cells in 75 cm² tissue culture flasks in a humidified incubator with 5% CO₂ at 37 °C, using the preferred culture medium for the cell type in question. Allow the cells to grow until they reach a near-confluent cell layer.        
   NOTE: Use Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) for LNCaP, HEK293, mouse embryonic fibroblasts (MEFs), BJ, MCF-7, and RPE-1 cells.
   1. Wash the cells with 3 mL 37 °C phosphate-buffered saline (PBS), pH 7.4. Replace the PBS with 3 mL 0.25% (w/v) Trypsin-ethylenediaminetetraacetate (EDTA), and incubate the flask in a humidified incubator with 5% CO₂ at 37 °C until the cells detach (2–5 min).
   2. Resuspend the detached cells with a 7 mL culture medium containing 10% FBS. Mix a 10 µL cell suspension aliquot with 10 µL 0.4% Trypan Blue in a microcentrifuge tube, using a 0.5–20 µL pipette tip. Use the same pipette tip to immediately fill a counting chamber slide, and count the cells in an automated cell counter.
2. Prepare a suitable dilution (see note below) of the cell suspension from step 1.1.2 using a culture medium containing 10% FBS, and seed 1 mL of the diluted cell suspension in each well of a 12-well tissue culture plate (surface area ~3.8 cm²) using aseptic technique. Allow growth in a humidified incubator with 5% CO₂ at 37 °C until the desired cell density has been reached, e.g., 60–90% confluence at harvest.         
   NOTE: The appropriate dilution of the cell suspension that will give the desired cell confluency at harvest will vary from cell type to cell type, as well as according to the duration and type of experimental treatments. Thus, this must be empirically assessed in each case.
   1. For experiments that are to be both treated and harvested 2 days after seeding, seed 2.5 x 10⁵ LNCaP, HEK293, or MCF-7 cells, 5 x 10⁴ MEFs, 4 x 10⁵ BJ, or 1.5 x 10⁵ RPE-1 cells in each well of the 12-well plate.
   2. For cells that adhere loosely, coat the plates with the type of coating recommended for the cell type in question. For LNCaP (and HEK293) cells use plates coated with poly-D-lysine (PDL).
   3. To that end, add 500 µL PDL at 2.5 µg/mL in sterile H₂O to each well, and incubate the plates in a sterile environment for 30 min at room temperature (20–25 °C). Remove the PDL with suction, and wash each well briefly with 1 mL sterile H₂O.       
      NOTE: Generally, step 1.2.1 is done without any experimental treatments. However, if performing RNAi, it may be convenient to start a reverse transfection with the seeding.
3. Perform experimental treatments in duplicate or triplicate wells per condition.
   1. For example, treat the cells with 50 nM of the mTOR-inhibitor Torin1, which generally is an efficient inducer of autophagic sequestration, or subject the cells to acute serum- and amino acid starvation by washing the cells with 1 mL of amino acid-free Earle's Balanced Salt Solution (EBSS) medium, and subsequently incubate the cells in 1 mL of EBSS in a humidified incubator with 5% carbon dioxide (CO₂) at 37 °C.
   2. Leave one set of wells untreated in order to define background levels of sedimentable lactate dehydrogenase (LDH).
   3. Add a saturating amount of the post-sequestration inhibitor bafilomycin A1 (Baf) in the absence or presence of the experimental treatments, 3–4 h before cell harvest. Incubate the cells in a humidified incubator with 5% CO₂ at 37 °C.
      1. Use 100 nM Baf for LNCaP, HEK293, BJ, MCF-7, and RPE-1 cells, and 10 nM Baf for MEFs.
      2. For experimental treatments that have a duration of only 3–4 h (like those exemplified in step 1.3.1 typically have), add Baf simultaneously with the treatments. For longer experimental treatments, wait until 3-4 h before harvest, and add 2 µL of a 500x concentrated Baf stock directly into the medium.
      3. Mix by agitating the plate immediately after the addition of Baf. At this point, it is also recommendable to add macroautophagic sequestration inhibitors as controls, e.g., 10 mM of the pan-phosphoinositide 3-kinase (PI3K) inhibitor 3-methyl adenine (3MA), or 10 µM of the selective PI3K class III inhibitor SAR-405.

2. Cell Harvest and Preparation for Electrodisruption

1. At the end of the treatment period, aspirate the medium with suction and add 200 µL cell detachment solution (pre-heated to 37 °C) to each well. Incubate at 37 °C until the cells detach (typically around 5 min).    
   NOTE: Whereas 0.25% (w/v) Trypsin-EDTA may be used instead of the cell detachment solution, the latter contains DNase, which helps reduce the viscosity of the detached cells. As long as the medium is thoroughly aspirated, it is not necessary to wash the cells before the addition of Trypsin-EDTA or cell detachment solution.
2. Add 500 µL room temperature (20-25 °C) PBS, pH 7.4, containing 2% (w/v) bovine serum albumin (BSA) to each well, and resuspend with the pipette until no cell clumps are visible. Immediately transfer the cell suspension to 1.5 mL microcentrifuge tubes on ice.      
   NOTE: Unless otherwise stated, perform all subsequent steps on ice.
3. Sediment the cells by centrifugation at 400 x g for 5 min at 4 °C.
4. Thoroughly aspirate the supernatant (with suction) to leave the cell pellets as dry as possible.
5. Add 400 µL 10% (w/v) sucrose (in ultrapure H₂O) to each tube.

3. Plasma Membrane Electrodisruption and Separation of Sedimentable- and Total-cell Fractions

1. Resuspend the cell pellet with a pipette to obtain a near single-cell suspension, and transfer it to a 4 mm electroporation cuvette.         
   NOTE: Pipetting up and down ~10–15 times, using a 100–1,000 µL pipette tip, is usually sufficient.
2. Place the cuvette in an exponential decay wave electroporator, and discharge a single electric pulse at 800 V, 25 µF, and 400 Ω; these settings produce a pulse of ~8 ms duration.
3. Use a new pipette tip to transfer the cell disruptate to a 1.5 mL microcentrifuge tube containing 400 µL ice-cold phosphate-buffered sucrose solution (100 mM sodium monophosphate, 2 mM dithiothreitol (DTT), 2 mM EDTA, and 1.75% sucrose, pH 7.5), and mix briefly by pipetting.
   1. Optional: To verify efficient plasma membrane electro-disruption, mix 10 µL of the diluted cell disruptate from step 3.3 with 10 µL 0.4% Trypan Blue in a 1.5 mL microcentrifuge tube. Transfer to a counting chamber and verify that the percentage of Trypan Blue positive cells is >99%.
      1. Leave the sample in the counting chamber for 30 min at room temperature (20–25 °C), and verify that the percentage of Trypan Blue positive cells has remained >99%.
   2. Optional: To verify that the electro-disruption has not been too harsh, that is, it has not disrupted intracellular organelles, perform steps 1.1–3.3 as described above, but use a larger starting material (a well from a 6-well plate with an ~80% confluent cell layer), and use 150 µL 10% sucrose in step 2.5 and 150 µL phosphate-buffered sucrose solution without DTT in step 3.3.
      1. Use a pipette to carefully layer 200 µL of the diluted cell disruptate solution on top of a 1.2 mL density cushion of phosphate-buffered 8% (w/v) density gradient medium (e.g, 8% Nycodenz, 50 mM sodium phosphate, 2.2% sucrose, 1 mM EDTA) in a 2 mL centrifuge tube. Centrifuge at 20,000 x g for 45 min at 4 °C in a microcentrifuge with soft-mode function (for gentle acceleration and deceleration), and carefully put the tubes on ice.
      2. Carefully remove 60 µL of the ~200 µL top fraction, making sure not to pick up any density gradient medium solution, and transfer to a fresh microcentrifuge tube.        
         NOTE: This should contain cytosol of exceptional purity, termed "cell sap".
      3. Test the purity of the fraction obtained in the above step, by performing western blot analyses of organelle-contained proteins, using standard techniques and 4–20% gradient gels.
      4. Perform for example immunoblotting for cathepsin B, cytochrome c, and protein disulfide isomerase, to verify that the electric shock in step 3.2 has not disrupted lysosomes, mitochondria, or endoplasmic reticulum, respectively, and immunoblot for LDH to verify the presence of a cytosolic protein in the cell sap.
      5. In parallel, perform immunoblotting on protein extracts made from the total cell disruptate solution to confirm that the antibodies used can detect the organelle-contained proteins that are being assessed.
4. Repeat steps 3.1–3.3 for each sample.
5. Remove 550 µL from each diluted cell disruptate solution (obtained at step 3.3) to 2 mL microcentrifuge tubes containing 900 µL ice-cold resuspension buffer (50 mM sodium monophosphate, 1 mM DTT, 1 mM EDTA, and 5.9% sucrose, pH 7.5) supplemented with 0.5% BSA and 0.01% Tween-20, and mix briefly by pipetting.
6. Centrifuge at 18,000 x g for 45 min at 4 °C to produce pellets containing "sedimented LDH". Thoroughly aspirate the supernatant (with suction) to leave the pellets as dry as possible. Place the samples in a -80 °C freezer.
7. Transfer 150 µL from each diluted cell disruptate solution (obtained at step 3.3) to new tubes, and place the samples in a -80 °C freezer. Use these samples to determine the "total LDH" levels in the cells.   
   NOTE: At this point, the experiment can be paused for as long as desired.

4. LDH Extraction and Measurement of LDH Enzymatic Activity

1. Thaw the "sedimented LDH" (from step 3.6) and "total LDH" samples (from step 3.7) on ice.
2. Add 300 µL of ice-cold resuspension buffer containing 1.5% Triton X-405 to the "total LDH" samples (yielding a final Triton X-405 concentration of 1%). Rotate the samples on a roller in a cold room (4–8 °C) for 30 min.
3. Add 750 µL of ice-cold resuspension buffer with 1% Triton X-405 to the "sedimented LDH" samples, and resuspend the pellets with a pipette until a homogenous solution is reached.
4. Centrifuge the samples from steps 4.2 and 4.3 at 18,000 x g for 5 min at 4 °C to sediment undissolved cellular debris.
5. Mix 4 parts of cold 65 mM imidazole (pH 7.5)/0.75 mM pyruvate with one part of cold 65 mM imidazole (pH 7.5)/1.8 mM NADH to obtain a working solution that is stable for at least three weeks at 4 °C.
6. Mix 3–30 µL of the supernatants from step 4.4 with 200 µL of the step 4.5 working solution.
7. Determine the amount of LDH by measuring LDH enzymatic activity as the decline in nicotinamide adenine dinucleotide (reduced form) (NADH) absorbance at 340 nm at 37 °C compared to a standard with a known LDH concentration. Perform absorbance measurements until the reaction has approached completion, i.e. until the absorbance at 340 nm no longer changes with time. 
   NOTE: This is the classical biochemical method to measure LDH activity. Although the current protocol performs the reaction at 37 °C, it can also be performed at room temperature (20–25 °C), which is advisable if doing manual spectrophotometry. The current protocol uses a robotic multi-analyzer instrument, which in an automated fashion mixes samples with working solution in a 96-well plate, and measures the absorbance at 340 nm at 37 °C every 20 s for 3 min. Thereafter, the instrument software calculates the concentration of LDH, expressed as Units (U)/L, by comparing the slope of the absorbance measurements over time compared to a standard curve obtained through calibration with a standard of known LDH concentration. The linear range of detection by this approach is 30–1,500 U/L. As an alternative, a wide variety of commercially available kits to measure LDH exist. Some of them are based on coupling the enzymatic reaction to the generation of colorimetric or fluorescent products, enabling detection by other means than UV spectrophotometry, and with other linear ranges of detection.