Karin,
I'm not sure your stance on metrizamide is correct.
Metrizamide strongly resembles the thyroid hormones T3 and T4, and their
breakdown product T2, as well as parts of iodinated thyroid globulin. We
know that for macrophages and dendritic cells, T3 is a strong activation and
differentiation agent. This became quite significant as more labs began to
use metrizamide for cell enrichment (alla Stella Knight). Hemmo Drexhage has
published several papers looking at the effects of this compound on monocyte
and macrophage activation. Here are several references:
Mooij P, Simons PJ, de Haan-Meulman M, de Wit HJ, Drexhage HA.
Related Articles
Effect of thyroid hormones and other iodinated compounds on the transition
of monocytes into veiled/dendritic cells: role of granulocyte-macrophage
colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6.
J Endocrinol. 1994 Mar;140(3):503-12.
Mooij P, de Haan-Meulman M, de Wit HJ, Drexhage HA. Related Articles
Thyroid hormones and their iodinated breakdown products enhance the
capability of monocytes to mature into veiled cells. Blocking effects of
alpha-GM-CSF.
Adv Exp Med Biol. 1993;329:633-6.
Kabel PJ, de Haan-Meulman M, Voorbij HA, Kleingeld M, Knol EF, Drexhage
HA. Related Articles
Accessory cells with a morphology and marker pattern of dendritic cells can
be obtained from elutriator-purified blood monocyte fractions. An enhancing
effect of metrizamide in this differentiation.
Immunobiology. 1989 Oct;179(4-5):395-41.
Whether the same holds true for other leukocytes, I don't know, but I would
perform a lot of preliminary tests before using metrizamide.
As for Ficoll, as long as you purchase an endotoxin tested product, or
perform endotoxin testing on your own, there is little chance of these
sucrose solutions activating your cells. Do a long term incubation with
graded doses of Ficoll at 37 degrees and measure upregulation of CD69. If
you see no upregulation of CD69 after 6 hours at 37 degrees, I doubt you'll
activate the cells in the 15-20 minutes it takes to run the gradient.
--
Science is built with facts as a house is with stones--but a collection of
facts is no more a science than a heap of stones is a house. -Jules Henry
Poincare (1854-1912)
Keith Bahjat
Graduate Assistant
University of Florida
College of Medicine
Gainesville, Florida
Phone: (352) 392-4887
Fax: (352) 392-5393
kbahjat@ufl.edu
on 11/16/00 05:46, K Haverson, School of Clinical Veterinary Science at
Karin.Haverson@bristol.ac.uk wrote:
>
> Dear Rick,
> Try using a metrizamide gradient, this is supposed not to activate
> cells. Our recipe:
> 18% metrizamide gradient. Prepared as follows:
> 1) Stock of 35.3% metrizamide w/v into distilled water.
> 2)Working soloution: 1.02ml stock + 0.94ml of PBS +0.04% Fetal calf
> serum. Prepare 1ml of 18% metrizamide for 1ml of cell
> suspension, at 1*107 cells /ml.
> 3)Underlay cells with metrizamide, spin at RT, 1500 rpm for 15mins, no
> brake.
> Karin Haverson
>
> On Tue, 14 Nov 2000 18:04:26 -0500 "Richard K. Meister"
> <meister.1@osu.edu> wrote:
>
>> Hello, everyone:
>>
>> One of our graduate students is trying to develop an assay for enumerating
>> mouse splenocytes which are activated in response to a particular antigen.
>> He is using anti-IFN-gamma production to identify activated cells. His
>> protocol requires approximately 1 week in cell culture, during which time
>> most non-activated splenocytes die (a majority of the starting population
>> of cells). This is followed by a short period of restimulation with the
>> antigen and staining for IFN-gamma.
>>
>> Flow cytometry analysis is hampered by the large population of dead cells
>> (not to mention that the dead cells are probably soaking up his monoclonal
>> antibody during staining). He is reluctant to use a Ficoll-Hypaque
>> gradient to clean up the dead cells for fear that this would result in
>> non-specific activation of his cells.
>>
>> Do any of you have a method of cleaning up the dead cells prior to cell
>> staining that would not result in activation of the viable cells?
>>
>> Thanks in advance,
>>
>> Rick Meister
>> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
>> * Richard K. Meister Email: meister.1@osu.edu *
>> * The Ohio State University Voice: (614) 292-9716 *
>> * Dept. of Veterinary Biosciences FAX: (614) 292-6473 *
>> * Cytometry Instrumentation Lab *
>> * 1925 Coffey Road *
>> * Columbus, OH 43210 U.S.A. *
>> * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
>>
>
> ----------------------
> Dr.K Haverson,
> Division of Clinical Veterinary Science,
> University of Bristol, Langford,
> BS40 5DU, UK
> e-mail: Karin.Haverson@bristol.ac.uk
> Tel.: (44) 117 928 9289
> FAX: (44) 117 928 9505
>
>
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