Hi All,
We have decided to isolate endothelial cells from our mouse model and I am in the process of learning and establishing a protocol so i have a few questions:
Other than my questions, I will really appreciate and be greatful for any other tips and considerations you can offer.
Maayan
Maayan,
Magnetic bead technology is very useful. Magnetic beads vary in size and composition and some cells "ingest" them or are otherwise activated or poisoned by them. Two strong personal opinions: (1) Purchasing high quality commercially available cells is nearly always more cost effective and reproducible than "home-made". (2) Never heat-inactivate serum in your own laboratory. If you feel there is a scientific basis to do so, ( I have seen none since the 1980's) buy it from a commercial laboratory that can create this reagent properly.
Please update us
Michael
Thank you Michael,
Regarding buying commercially available cells - that is not a possibilty, there is a very specific mutation we introduced into our mice that we work on, I need to isolate the cells for this reason and to further understand the phenotype we see in vivo.
Thank you for the serum tip, I will bear it in mind.
Hi,
1. Magnetic bead separation: We have used the Miltenyi AutoMACS for cell separation on primary cells for the last 8 years and did it manually with their system before that (I have used the dyna bead system in the past though). Our main uses are isolating EPCs and late outgrowth endothelial cells from human peripheral or cord blood or bone marrow and ECs from mouse bone marrow but we have played with a number of things over the years. I know none really wants to buy in something else to do the job is but what we've found that has become invaluable is the ability to precisely control your sort. for example you can sort with cell numbers as the priority so you might get some cell contamination or vice versa you might want a high purity cell sort but at the cost of cell numbers. this was important as we found for our CD133 sorts for rare cells in the blood we wanted as many cells as possible and the cells would actually grow better with a little "contamination" of non CD133 cells. additionally for our neutrophil sorts it mattered about if the sort was done as a selection or depletion and the sort parameters were altered accordingly.
2. freezing cells: we have frozen HUVEC, VERAVEC, late outgrowth Endothelial cells, MNCs and mouse endothelial cells all with no worries. even fresh CD34 or CD133 sorted cells from blood freeze fine for future culture or experiments. the one issue we had was freezing mononuclear cells and then thawing them and needing to sort. there's so many dead cells that stick to beads the sort would be rubbish. we tried the dead cell removal kit from miltenyi which also consists as a sort and it does work but it is very cost prohibitive so we just did the sort and freeze.
3. Trypsin: we use 0.05% with all our endothelial lines and never had any issues with PECAM staining straight after. I have heard TripLE (invitrogen) is a good alternative though
hope there's something useful to you in the above,
Goodluck!!
Thank you Michaelia,
we are intending to use the cells for migration and permeability assays and not for further sorting but thank you very much for the tips and insights, they are very helpful.
Maayan
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