Greetings and thanks for the responses to my question posted Friday,
20-April!!
I have been using EDTA removal for years but had reservations about these
cells and Macrophages in general. See below:
>I have been engaged to examine surface markers on PMA- treated THP-1
> cells by flow. I expect the cell-removal process to be difficult,
> and while there are references to be found which include such cytometric
data, the
> details on cell harvest have been sketchy.
Response 1:
"Hey Sam, we've analyzed these cells for surface proteins without difficulty
after
removal with EDTA. There was a post in recent days on keratinocytes
describing an EDTA concentration and other useful tips - like the cells
are easier to detach if they aren't grown to confluency, etc.. The EDTA
concentration in the keratinocyte posting seemed low compared to what
we were using. I think we used 10 mMolar EDTA in PBS to detach. Cells
should
detach within a few minutes, 3-5. If it takes much longer you'll run into
viability problems."
Response 2:
"Collagenase, Keeping cells on ice are the two things that come to my mind."
Response 3:
"We have done this in several cell lines as well as primary cells. We
always use EDTA, from 1mM to 10mM, depending on the "attachedness" of the
cells.
Here is a rough protocol:
1. Remove media from attached cells
2. Wash with PBS (or HBSS, and so throughout)
3. Add small amount of PBS with 1-10 mM EDTA ("amount" corresponds to the
dish or flask size, for a T25 use 1.5 ml; for a well in a 24-well dish,
use 100 microliters)
4. Incubate up to 15 minutes while monitoring by microscope for "rounding
up" of cells **some folks put the cells on ice to also dislodge them if
difficult.
5. When you can see the cells are "rounded up," then you can either
pipette vigorously with a P1000, or scrape them off with sterile cell
scrapers.
6. Collect cells into new, fresh media to remove effects of EDTA (and to
dilute the EDTA out), then centrifuge the cells out of EDTA-media. Many
binding studies require the removal of EDTA for appropriate binding
assays."
(SW) When you say primary cells, do you mean macrophages?
"Yes, from peripheral blood. We have differentiated them in vitro for 7-10
days with cytokines or infectious agents, and *then* removed them for flow."
Thanks again to
Julie, Tom, and Padma
Cheers,
Sam
Sam Witherspoon
sw11527@gsk.com
Tel. 919-483-3078
GlaxoSmithKline R&D Page 919-857-7768
5 Moore Dr. Fax 919-483-0585
RTP, NC 27709
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