CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
It is a cationic amphiphilic tracer (CAT) that selectively labels autophagic vacuoles	Treatment of BMDM on coverslips with C9433 (rocaglate) detects autophagic vessicles using this kit in FITC channel

Upstream tips
It is a cationic amphiphilic tracer (CAT) that selectively labels autophagic vacuoles
Protocol tips
Treatment of BMDM on coverslips with C9433 (rocaglate) detects autophagic vessicles using this kit in FITC channel

Publication protocol

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilised with 0.25% Triton-X for 30 min and then blocked for 20 min with goat-serum (2.5%). Cells were incubated with primary antibodies [mouse monoclonal antibodies against Ipr1(1:2000), iNOS(1:200) and LC3B(1:250)] overnight at 4 °C in 2.5% goat serum, and incubated with Alexa Fluor 594-conjugated donkey anti-mouse IgG (excitation/emission maxima ~ 590/617 nm) or Alexa Fluor 488- conjugated donkey anti-mouse IgG (excitation/emission maxima ~ 490/525 nm) (1:1000, Invitrogen) secondary antibody for 2 hrs. F.t. LVS were detected with anti-mouse F.t. LVS antibodies (1:1000). BMDMs from C57BL/6 J mice were grown in coverslips and treated with compounds with indicated time and autophagy was detected using the cyto-ID Autophagy detection kit (Enzo lifesciences) using FITC channel. Images were taken immediately in Nikon-deconvolution microscope. To detect autophagy in immortalized GFP-LC3 + (ex/emi max ~488/510 nm), iBMM was grown as mentioned previously65 and samples were processed as mentioned above. Images were acquired using Leica SP5 confocal microscope. All images were processed using Image J software.

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

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