I use beads conjugated with anti-CD14 in PBMCs samples in order to purify monocytes through a column linked to a magnetic board, which is obtained from MACS. However, using this kind of kit is too expensive. In this sense, I would like to know if there is an alternative way to obtain them from PBMCs.
It was suggested to me by a researcher from another lab for incubating PBMCs in 24-well for 2 hours, because through this methodology, monocytes would attach to the bottom and, then, I could make some washes in order to discard other cells left onto there, and, in this way, to obtain the monocytes that were attached. I am afraid though of losing a lot of cells by this process.
Dear Amanda Rocha,
I agree with the idea of letting monocytes to adhere and wash other non-adherent cells. This is the regular practice most of the lab follows. I also agree with your concern that you may lose some cells but not lot of cells. The fact is that monocytes are very low in number in PBMCs compared to lymphocytes and PMNs. These lymphocytes and PMNs are non-adherent cells. So when you wash away non-adherent cells you remain with only monocytes which are low in number and by looking at large number of non-adherent cells you might think that you are losing a lot of monocytes.
Hello Amanda
I advise you to isolate monocytes by its adhesive property. First at all, By differential gradient centrifugation by ficoll obtaining mononuclear cells from PBL (Interface). Then, calculate the cell density of monocyte respect to total PBL (approximately 10%) and seeding. After 24 hours in incubation time, the adherence cells are monocytes and the rest of cells are lymphocytes mainly. Please, check this paper.
Best regards
https://www.ncbi.nlm.nih.gov/pubmed/26800307
By using the adherence property to plastic surfaces of monocytes, the loss of certain cell is inevitable. In any case, the process can be improved with some heed. After separation, re-suspend PBMCs in PBS+ FSC 10% in a Petri's plate for 4h. Then, discard the supernatant. The monocytes can be detached by subsequent washes with warm PBS+FCS 10% (37C). During the last washing a spatula can be used gently.
Hi Amanda,
you can use a density gradient with lower density, 1.68-1.70 will reduce the contamination with other t-,b and NK cells. Additional you have to be careful not to overload you cell culture dish, so you monocytes will find space to adhere. There will be a small problems with platelets since this also adhere to the cell culture plate. This you could minimize with a platelet depletion Kit (https://pluriselect.com/product-details/product/plurispin-human-plt-depletion.html).
Good luck Michael
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts