Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells

Abstract

Source: Luhr, M. et al., The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells. J. Vis. Exp. (2018)

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....

Materials

List of materials used in this article

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