Autophagy assay cell type - THP 1

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 16 matching solutions for this experiment

Atg5 (D5F5U) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’	Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Upstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’
Protocol tips
Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’	Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Upstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’
Protocol tips
Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

Atg12 (D88H11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’	Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Upstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’
Protocol tips
Primary antibody is together treated with protein A/G Plus-agarose immunoprecipitation reagent at 4 °C for 3 h or overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B Antibody

Novus Biologicals

Upstream tips	Protocol tips	Downstream tips
Lyse cells in uffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors	Dilute primary Ab at 1:5,000 and incubate overnight at 4C in a humidified container

Upstream tips
Lyse cells in uffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitors
Protocol tips
Dilute primary Ab at 1:5,000 and incubate overnight at 4C in a humidified container

Manufacturer protocol Publication protocol 1 Relevant paper

SAPK/JNK Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in in a modified RIPA buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Beclin-1 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Purified Mouse Anti-p62 Ick ligand Clone 3/P62 LCK LIGAND (RUO)

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors	Incubate primary Ab overnight at 4C.

Upstream tips
Lyse cells in buffer containing 150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid, 0.1% SDS 1% Triton X-100, protease and phosphatase inhibitors
Protocol tips
Incubate primary Ab overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Purified Mouse Anti-Beclin Clone 20/Beclin (RUO)

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
Lyse cells in buffer containing 0.125 mol/L Tris, pH 6.8, 4% SDS, 20% glycero, 10% 2 mercaptoethanol	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Upstream tips
Lyse cells in buffer containing 0.125 mol/L Tris, pH 6.8, 4% SDS, 20% glycero, 10% 2 mercaptoethanol
Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
	Dilute 4 μl of Cyto-ID® Green Detection Reagent in 1ml of diluted assay buffer and stain the cells for 30 min at 37°C in the dark.

Protocol tips
Dilute 4 μl of Cyto-ID® Green Detection Reagent in 1ml of diluted assay buffer and stain the cells for 30 min at 37°C in the dark.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3B Antibody

Novus Biologicals

Upstream tips	Protocol tips	Downstream tips
	For WB dilute primary Ab at 1:1000 and for IF at 1:200 and incubate at 4°C with gentle shaking, overnight.

Protocol tips
For WB dilute primary Ab at 1:1000 and for IF at 1:200 and incubate at 4°C with gentle shaking, overnight.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	For immunostaining, incubate cells with primary Ab overnight at 4°C

Protocol tips
For immunostaining, incubate cells with primary Ab overnight at 4°C

Manufacturer protocol Publication protocol 1 Relevant paper

SQSTM1/p62 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Atg3 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

SQSTM1/p62 (D5E2) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

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