EVs were collected from 50mL of MIA PaCa-2 cells supernatant cultured for 72h in RPMI medium supplemented with EVs-depleted FBS. EVs isolation was performed as previously described. Then, EVs pellet was resuspended in 100μL of PBS 1X and divided into four conditions for the analysis of CD63, CD81, CD82 and Rab5. A similar protocol to the one performed for the analysis of Evs by flow cytometry was done until detection in ImageStream× MarkII Imaging Flow Citometry (Luminex Amnis Image Stream Multispectral Imaging Flow Cytometer, RRID:SCR_018589). Primary antibodies used were: anti-CD63 1:400 (Santa Cruz Biotechnology Cat# sc-15363, RRID:AB_648179), anti-CD81 1:400 (Abcam Cat# ab109201, RRID:AB_10866464), anti-CD82 1:400 (Abcam Cat# ab59509, RRID:AB_2076398) and anti-Rab5 1:400 (Cell Signaling Technology Cat# 46449, RRID:AB_2799303). Secondary antibodies used were: Donkey anti-mouse IgG Alexa Fluor 488 1:500 (Molecular Probes Cat# A-21202, RRID:AB_141607) and Goat anti-rabbit IgG Alexa Fluor 488 1:500 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217). For each sample, control refers to EVs incubated with secondary antibody only.
Ab59509
Manufactured by Cell Signaling Technology
Ab59509 is a primary antibody product offered by Cell Signaling Technology. It is designed for use in various immunological techniques.
Automatically generated - may contain errors
2 protocols using ab59509
1
Quantification of EV Markers in MIA PaCa-2 Cells
2
Imaging Flow Cytometry of EV Markers
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EVs were collected from 50 mL of MIA PaCa-2 cell supernatant cultured for 72 hours in RPMI medium supplemented with EV-depleted FBS. EV isolation was performed as previously described. Then, EV pellet was resuspended in 100 µL of PBS 1× and divided into four conditions for the analysis of CD63, CD81, CD82 and Rab5. A similar protocol to the one performed for the analysis of Evs by flow cytometry was done until detection in ImageStreamx MarkII Imaging Flow Citometry (Luminex Amnis Image Stream Multispectral Imaging Flow Cytometer, RRID:SCR_018589). Primary antibodies used were anti-CD63 1:400 (Santa Cruz Biotechnology Cat# sc-15363, RRID:AB_648179), anti-CD81 1:400 (Abcam Cat# ab109201, RRID:AB_10866464), anti-CD82 1:400 (Abcam Cat# ab59509, RRID:AB_2076398) and anti-Rab5 1:400 (Cell Signalling Technology Cat# 46449, RRID:AB_2799303). Secondary antibodies used were Donkey antimouse IgG Alexa Fluor 488 1:500 (Molecular Probes Cat# A-21202, RRID:AB_141607) and goat antirabbit IgG Alexa Fluor 488 1:500 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217). For each sample, control refers to EVs incubated with secondary antibody only.
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