|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
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 Full paper
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