Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells
Published: January 31, 2024
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 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, …
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
Bu Makaleden Alıntı Yapın
Quantifying Bulk Autophagic Sequestration Activity in Mammalian Cells. J. Vis. Exp. (Pending Publication), e21925, doi: (2024).