Shreffler:Notebook/Staff Meetings/2010/05/07

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] ShreffLab Staff Meetings
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Agenda Items

 * look up GI fellow working in Mucosal Immunology to put in touch with Bert

Cytokine mRNA levels
 * ALDH variation -- fresh cells
 * Bert's conclusions:
 * ALDH1A2 was upregulated by CPE in mDC, but not as much as I've seen before at Mt Sinai. This probably explains the small effect of CPE on RA-sensitive T cell surface markers.
 * No difference between these fresh cells and recent results with buffy coats. Therefore, low response to CPE is probably not due to buffy coat production.
 * Could be the medium. Changed to new batch around the same time as when I had the one good experiment (done on 3/12/10, stained T cells on 3/18/10). So could be due to new lot of medium, or to the Pen/Strep, which is also different from the Pen/Strep used at Mt Sinai.
 * Will test various media and Pen/Strep variants in culture with mDC and use ALDH1A2 expression/upregulation by CPE as readout.
 * Will also test lots of stem cell media

The cells were cultured for 22h and 46h. Results were compared with previous exp, in which 10 nM RA was used.

- IL-5 is upregulated by RA under various conditions of T cell stimulation. Use of anti-CD3 beads in 1:30 gives the highest IL-5 upregulation without much effect on the other measured cytokines (IL-4, IL-13, IFN-g).

- Stimulation with 1 pg/ml SEB also gives a modest IL-5 response.

- Stimulation with anti-CD3 beads in 1:120 upregulates all four cytokines after ~45h.

[[Media:QPCR4-15-10.xls]]

[[Media:QPCR5-5-10A.xls]]

[[Media:QPCR5-5-10B.xls]]

[[Media:SummarycytokineQPCR.xls]]

Cytokine CBA data will follow.

Previous Action Items

 * Purification
 * Columns are in, Bert has been prioritizing the DC comparisons; may shift now some to purification as we need to get material to off site
 * access to speed vac -- unresolved, but may be part of what 3rd party can do
 * look back to compare qPCR and T cell readouts
 * Bert will look at -- hopefully this week
 * Kaye has sent protocol details to Steve
 * Wayne will follow up with him to find out if he has any suggestions/ insight