If titre is positive do one of the following 2 weeks later:
Maintenance of the cells:
Seed macrophages at a density of 1.5x105 cells/ml in the medium described on next page. Add 2.5 ug/ml LPS which induces differentiation.
Collect sup after 2-3 days, or when medium is getting too yellow. Induce 2 more times , each time with 1 ug/ml LPS and collect sup after 2 days each.
Pool the sups, filter and use as recommended. (Could be aliquoted and stored at -20C.)
Preparation of Media:
for P3X63-Ag8.653: IMDM complete to 425 ml of IMDM add:
for fox-NY: IMDM or RPMI
for J774A.1: same medium as the one for the P3X63-Ag8.653 ( = Ag8 ).
The Macrophages optimal density is 1.5x105/ml. When expanding them , use a "policeman" to scrape them; this is easier to do if they are growing in petri dishes at this stage.
Freezing Hybridoma / Myeloma / macrophages.
Freezing solution: 90% FCS + 10% DMSO, ice cold.
|Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: email@example.com |
David Bowtell PMCI October 1998