Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Overview

The video demonstrates an assay for assessing bulk autophagic sequestration activity in mammalian cells. It utilizes electroporation to release cytosolic proteins, including lactate dehydrogenase (LDH). The sequestered LDH and total cellular LDH fractions are determined to assess LDH sequestration, providing insights into the overall bulk sequestration activity.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
1.5 ml and 2 ml microcentrifuge tubes	Eppendorf	211-2130 and 211-2120
12-well plates	Falcon	353043
Accumax cell detachment solution	Innovative Cell Technologies	A7089	Keep aliquots at -20 °C for years, and in fridge for a few months
Bafilomycin A1	Enzo	BML-CM110-0100	Dissolve in DMSO
BJ cells	ATCC	CRL-2522	Use at passage <30
Bovine serum albumin (BSA)	VWR	422361V
Burker counting chamber	Fisher Scientific	139-658585
Countess Cell Counting Chamber Slides	ThermoFisher Scientfic	C10228
Countess II Automated Cell Counter	ThermoFisher Scientfic	AMQAX1000
Cover glass for the Burker counting chamber	Fisher Scientific	139-658586
Criterion Tris-HCl Gel, 4–20%, 26-well, 15 µl, 13.3 x 8.7 cm (W x L)	Bio-Rad	3450034
DTT	Sigma-Aldrich	D0632
Earle's balanced salt solution (EBSS)	Gibco	24010-043	Conatains 0.1% glucose
EDTA	Sigma-Aldrich	E7889
Electroporation cuvette (4 mm)	Bio-Rad	1652088
Exponential decay wave electroporator	BTX Harvard Apparatus	EMC 630
Fetal bovine serum (FBS)	Sigma	F7524	10% final concentration in RPMI 1640 medium
HEK293 cells	ATCC	CRL-1573
Imidazole	Sigma-Aldrich	56750	Autoclave a 65 mM solution and keep in fridge for months
Incubator; Autoflow IR Direct Heat CO2 incubator	NuAire	NU-5510E
Lipofectamine RNAiMAX Transfection Reagent	ThermoFisher	13778150
LNCaP cells	ATCC	CRL-1740	Use at passage <30
3-Methyl Adenine (3MA)	Sigma-Aldrich	M9281	Stock 100 mM in RPMI in -20 °C.  Heat stock to 65 °C for 10 minutes, and use at 10 mM final concentration
Refridgerated Microcentrifuge	Beckman Coulter Life Sciences	368831
Refridgerated Microcentrifuge with soft-mode function	Eppendorf	Eppendorf 5417R
MRT67307 hydrochloride (ULKi)	Sigma-Aldrich	SML0702	Inhibits ULK kinase activity. Dissolve in DMSO.
MaxMat Multianalyzer instrument	Erba Diagnostics	PL-II
MCF7 cells	ATCC	HTB-22
NADH	Merck-Millipore	1.24644.001
Nycodenz	Axis-Shield	1002424
Opti-MEM Reduced Serum Medium	ThermoFisher	31985062
Phosphate-buffered saline (PBS)	Gibco	20012-019
Pipette tips 3 (0.5-20 µl)	VWR	732-2223	Thermo Fischer ART Barrier tips
Pipette tips (1-200 µl)	VWR	732-2207	Thermo Fischer ART Barrier tips
Pipette tips (100-1000µl)	VWR	732-2355	Thermo Fischer ART Barrier tips
Pipettes	ThermoFisher	4701070	Finnpipette F2 GLP Kit
Poly-D-lysine	Sigma-Aldrich	P6407-10X5MG	Make a 1 mg/ml stock solution in sterile H2O. This solution is stable at -20 °C for at least 1 year.
Pyruvate	Merck-Millipore	1066190050
RPE-1 cells (hTERT RPE-1)	ATCC	CRL-4000
RPMI 1640	Gibco	21875-037
SAR-405	ApexBio	A8883	Inhibits phosphoinositide 3-kinase class III (PIK3C3). Dissolve in DMSO.
Silencer Select Negative Control #1 (siCtrl)	ThermoFisher/Ambion	4390843
Silencer Select ATG9-targeting siRNA (siATG9A)	ThermoFisher/Ambion	s35504
Silencer Select FIP200-targeting siRNA (siFIP200)	ThermoFisher/Ambion	s18995
Silencer Select ULK1-targeting siRNA (siULK1)	ThermoFisher/Ambion	s15964
Silencer Select ULK2-targeting siRNA (siULK2)	ThermoFisher/Ambion	s18705
Silencer Select GABARAP-targeting siRNA (siGABARAP)	ThermoFisher/Ambion	s22362
Silencer Select GABARAPL1-targeting siRNA (siGABARAPL1)	ThermoFisher/Ambion	s24333
Silencer Select GABARAPL2-targeting siRNA (siGABARAPL2)	ThermoFisher/Ambion	s22387
Sodium phosphate monobasic dihydrate (NaH2PO4 • 2H2O)	Merck-Millipore	1.06580.1000
Sodium phosphate dibasic dihydrate (Na2HPO4 • 2H2O )	Prolabo	28014.291
Sucrose	VWR	443816T	10% final concentration in water; filter through 0.45 µm filter and keep in fridge for months
Thapsigargin	Sigma-Aldrich	T9033	Inhibits the SERCA ER Ca2+ pump. Dissolve in DMSO.
Triton X-405	Sigma-Aldrich	X405	1% final
Trypan Blue stain 0.4%	Molecular Probes	T10282
Trypsin-EDTA (0.25% w/v Trypsin)	Gibco	25200-056
Tween-20	Sigma-Aldrich	P2287	0.01% final