Hi,
If I in vitro restimulate CFSE-labelled OT II whole splenocytes (not the CD4+ cells) for 3-5 days in culture with OVA or ISQA peptide, would I see the proliferation of CD4+ cells with a panel CFSE/CD4?
Thanks
vincent
Hi, thanks for your replies. These mice are transgenic, so most of CD4+ cells should be responsive, that's why I thought about 5 days. I know an immune response is around 10-12 days. I could add another time points.
I will apply a flow cytometry panel including a live/dead dye of course.
thanks.
You can please follow the links below to obtain certain crucial informations:-
1. http://www.jimmunol.org/content/176/5/3037.full
2. http://www.jimmunol.org/content/182/1/193.full.pdf
3. http://www.pnas.org/content/suppl/2012/04/17/1205150109.DCSupplemental/pnas.201205150SI.pdf
Since you are using whole spleen cells, you should be able to see some proliferating CD4+ T cells as the accessory cells should be able to present the peptides to CD4+ T cells. This is a routine protocol, and the links provided by Sandip above should be helpful.
Just checking the color of medium, after 5 days, ISQA restimulated wells are yellow, OVA orange-ish, and medium only still red/pink. Tomorrow I'll do the FACS staining...thanks
Great, I can see CD4+ cells proliferation in response to ISQA but not OVA, certainly a question of dose.
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