"P.M. Newton" wrote:
>> Hello
> I am attempting to extract peripheral blood monocytes and then differentiate
> them into macrophages, maintaining the cells as macrophages in culture for as
> long as poss.
> I have reliable protocols for the ficoll extraction proceedure, and can get
> good yields of monocytes, but these remain attached to the tissue culture
> plastic for a maximum of 48 hours before floating off, which is no good to me
> (i need at least a week).
> Can anyone offer any tips on how to keep the cells adherent and
> differentiated?
> i am aware a dose of M-CSF or GM-CSF may do the trick, but if anyone has any
> other (preferably cheaper!!) suggestions they would be most appreciated.
>> thanks
> Phil Newton
> SBMB
> Uni of Leeds.
Coating the plates with Immunoglobulin may help although the Ig will
stick better to non tissue culture plastic than tissue culture
plates.
The method we have used for FcR panning on petri dishes is as follows.
1. Make 5mg/ml stock solution of human gamma globulin [Cappell
chromatographically purified immunoglobulin Cat no 0001-0910] by
disolving 100mg into 20ml of sterile ddH2O.
2. Add 5mls of Ig solution to 100mm bacteriological petri dishes to
cover the bottom of the plate.
3. Allow to sit for 5 minutes and transfer the solution to a second and
subsequent plates as necessary. The IgG solution should be kept sterile
and may be reused after storage at 4°C.
4. Wash the pans twice with PBS and add cells in RPMI 10% Human serum
medium. Unused pans may be stored after adding 5mls PBS for up to 5 days
at 4°C but are best used on the day of preparation.
5. Incubate cells on plates for 2 hours at 37°C in the incubator and
wash off non adherent cells with warm PBS.
The panned cells remain tightly adherent during subsequent culture
compared to plastic adherenet cells although we only culture for 24-36
hours.
--
Paul U Cameron
Research Fellow, Department of Microbiology and Immunology
University of Melbourne
