In our lab we routinely isolate monocytes from peripheral blood (by
Percoll). To put in my two cents, I will use the list of Mayumi to give my
opinion and experience:
- Traditionally take adherence cell:
The purity of these cell is often poor, the number per cm2 or well is
different from well to well (or cm2 to cm2) and the loss of monocytes is
very large.
- MACS, http://www.miltenyibiotec.com/index.php
We get very nice purities with the positive selection, often above 95% and
always over 90%. But a rather expensive method because Percoll works even
better and is less expensive.
- Hydrophobic culture bag, Cheryl Guyre, JIM 2002 "Advantages of
hydrophobic culture bags over flask for the generation of monocyte-derived
DC......." This article must be on the very recent JIM. I have the
in-press version.
No experience.
- Percoll: http://memorias.ioc.fiocruz.br/952/3879.html
In our hands the best solution. We perform this routinely and get purities
around 95% +/- 4%.
In short(from: Blood, Vol. 90 No. 5 (September 1), 1997: pp. 1920-1926,Human
Dendritic Cells Require Exogenous
Interleukin-12-Inducing Factors to Direct the Development of Naive T-Helper
Cells Toward the Th1 Phenotype. By Catharien M.U. Hilkens, Pawel Kalinski,
Mark de Boer, and Martien L. Kapsenberg)
Generation of dendritic cells and macrophages from peripheral blood (PB).
Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated
from freshly drawn PB by density centrifugation on Lymphoprep (Nycomed,
Torshov, Norway). Subsequently, PBMC were centrifuged on a Percoll
(Pharmacia, Uppsala, Sweden) gradient, consisting of three density layers
(1.076, 1.059 and 1.045 g/mL). The light density fraction, containing
predominantly monocytes, was seeded in 24 well-culture plates (Costar,
Cambridge, MA) at a density of 0.5 ? 106 cells/mL. After a 1-hour incubation
at 37?C, nonadherent cells were removed and adherent cells were cultured in
Iscove's modified Dulbecco's medium (IMDM; Life Technologies Ltd, Paisley,
UK) and 10% fetal calf serum (FCS; Hyclone, Logan, UT), supplemented with
granulocyte-macrophage colony-stimulating factor (GM-CSF ) (500 U/mL) to
obtain macrophages (M) or supplemented with GM-CSF and IL-4 (250 U/mL) to
obtain DC.13,14 Every 2 days
the media including the supplements were refreshed. After 6 days of culture,
DC and M were procured and extensively washed before use.
Other references, to give an idea about our research.:
@ Kalinski P, Schuitemaker JH, Hilkens CM, Kapsenberg ML. Prostaglandin E2
induces the final maturation of IL-12-deficient CD1a+CD83+ dendritic cells:
the levels of IL-12 are determined during the final dendritic cell
maturation and are resistant to further modulation. J Immunol. 1998 Sep
15;161(6):2804-9.
@ Kalinski P, Schuitemaker JH, Hilkens CM, Wierenga EA, Kapsenberg ML. Final
maturation of dendritic cells is associated with impaired responsiveness to
IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature
dendritic cells to produce IL-12 during the interaction with Th cells.
@ Kalinski P, Smits HH, Schuitemaker JH, Vieira PL, van Eijk M, de Jong EC,
Wierenga EA, Kapsenberg ML. IL-4 is a mediator of IL-12p70 induction by
human Th2 cells: reversal of polarized Th2 phenotype by dendritic cells. J
Immunol. 2000 Aug 15;165(4):1877-81.
@ de Jong EC, Vieira PL, Kalinski P, Schuitemaker JH, Tanaka Y, Wierenga EA,
Yazdanbakhsh M, Kapsenberg ML. Microbial compounds selectively induce Th1
cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse
th cell-polarizing signals. J Immunol. 2002 Feb 15;168(4):1704-9.
- Sorter: Purity must be the best, but seems it depends on an instrument
and setting technique.
Purity is often better, but as already stated by Mayumi, the monocytes are
'fragile' and a large percentage will die in the first day. And the majority
will be activated for sure.
My top 3 would be:
1. Percoll
2. Positive selection by MACS
3. Adherence
Success,
Joost
____________________________________________________
J.H.N.Schuitemaker
Laboratory Manager / Senior Research Technician
Cellular Immunology Group
Cell Biology & Histology
Academic Medical Center
P.O.box 22700
1100 DE Amsterdam
The Netherlands
Telephone +31 (0)20 5664960
FAX +31 (0)20 6974156
Internet: http://www.amc.nl
http://home.wanadoo.nl/flowcytometry
-----Original Message-----
From: Mayumi Kataoka (Temple U) [mailto:kataokam_2000@yahoo.com]
Sent: dinsdag 3 december 2002 1:47
To: cyto-inbox
Subject: Re: Monocytes and Macs, AND MORE
I recall that monocyte isolation has become a topic several times, but I
am not sure which is the best way. It maybe depends on the purpose of each
experiment.
Is anyone out there actually who has compared different methods
considering purity, damage (physically or functionally), or stimulation?
Here is the methods I have experienced and known of.
- Traditionally take adherence cell: 50-60% Monos, of course they are
stimulated, but the scatter plot (FSC vs SSC) is better (tighter) than
MACS isolated monos.
- MACS, http://www.miltenyibiotec.com/index.php
Positive selection < 88% (never reached 90%)
Negative selection, much lower purity (forgot it because it was not
useful for us)
Isolated monos sometimes show a spread-out, rough scatter plot on FACS.
They are stimulated, and perhaps damaged. They could not survive well in
culture (the culture was performed by a grad student, not by me. Please
comment if you succeeded).
- Hydrophobic culture bag, Cheryl Guyre, JIM 2002 "Advantages of
hydrophobic culture bags over flask for the generation of monocyte-derived
DC......." This article must be on the very recent JIM. I have the
in-press version. According to a co-author, it was better purity, less
stimulation, and softer adhesion.
- Parcoll: http://memorias.ioc.fiocruz.br/952/3879.html
I have not tried yet. It says 90% of purity. Anyone tried?
- Sorter: Purity must be the best, but seems it depends on an instrument
and setting technique.
Does anyone have data regarding the functional assays, recovery rate, % of
death on day 0,1,2....?
I appreciate any return.
Thank you,
Mayumi Kataoka
FACS Facility
Temple University
Philadelphia, PA, USA
--- Annette Byrne <AByrne@pcyc.com> wrote:
> Hi Flowers
>
> I'm looking for some advice on the best way of separating out the
> monocyte
> population from peripheral blood and also how does one go about
> activating
> them to become terminal macrophages ?.
>
> Also for those of you in the know, is this the best system to look at
> human
> Macs. To date I've been using PMA activated THP-1 cells but am trying to
> get
> a more " physiological" way of doing things
>
> many thanks as always
>
> Annette
>
> Annette Byrne PhD
> Scientist
> Pharmacyclics
> 995 E Arques Ave
> Sunnyvale
> CA 94085-4521
> USA
>
> Tel: 001-408-3283640
> Fax: 001-408-3283689
> e mail : AByrne@pcyc.com
> www.pharmacyclics.com
>
>
>
__________________________________________________
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