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

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Agenda Items

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

Cytokine mRNA levels

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



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