I am interested in examining the status of autophagy in peripheral monocytes from patients with neurodegenerative disorders such as Alzheimer's. We take around 5ml of blood from patients and are trying to examine autophagy using a dye that stains autophagosomes. First issue we are facing is that we do not get enough monocytes from PBMCs isolated from 5ml blood. We are taking the adhering population. We were able to acquire only 2000 events. Any comments or suggestions?
To extract PBMCs, human blood samples collected in EDTA vacutainers were diluted 1:1 with serum containing DMEM in Lymphoprep tubes and centrifuged for 10 minutes at 1100 x g (18-22°C), no brake. PBMCs formed a distinct band which was collected and diluted in complete DMEM, which was then centrifuged for 10 minutes at 500 x g. After removing the supernatant cell viability and numbers were assessed using Trypan Blue staining and then used for downstream assays or culture.You should have just enough cells to continue with Cyto ID kit.
As for autophagy staining, yes have used Cyto ID kit, but on adherent cells, not PBMCs. Straightforward, no problems. Perhaps cytospin your freshly stained PBMCs onto slide for confocal?
Thanks Karen!
How do you treat your monocytes (the adherent population) with cyto ID reagent? Do you initially culture your cells or directly stain it with cyto id reagent? Will you be able to share the protocol with us. And also how long do you treat with positive control (rapamycin).
We are actually trying to analyze by Flowcytometery.
We have grown epithelial cells on coverslips or on glass chamber slides, then fixed in paraformaldehyde and followed the Enzo manual protocol http://static.enzolifesciences.com/fileadmin/files/formula/ENZ-51031_Cyto-ID_Autophagy_Detection_Kit_Rev_3_0_June_2012.pdf
I would be tempted to continue straight from buffy coat and stain all PBMCs as per live cell protocol, fix and FACS. Rapamycin effect varies, you could try 4 hours, but I haven't used it in PBMCs, just epithelial cells and Jurkats.
