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
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.
Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

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.
Atg12 (D88H11) Rabbit mAb

Cell Signaling Technology

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.
LC3B Antibody

Novus Biologicals

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
SAPK/JNK Antibody

Cell Signaling Technology

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.
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.
Beclin-1 Antibody

Cell Signaling Technology

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.
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.
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.
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.
LC3B Antibody

Novus Biologicals

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.
Protocol tips
For immunostaining, incubate cells with primary Ab overnight at 4°C
SQSTM1/p62 Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.
Atg3 Antibody

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.
SQSTM1/p62 (D5E2) Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:1,000 and incubate overnight at 4C.
Beclin-1 (D40C5) Rabbit mAb

Cell Signaling Technology

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