DC Culture Protocol

DC Media: RPMI + 10% FCS + Pen + Glu
Cytokines: IL-4 @ 500U/ML
GMCSF @ ?
Plates: Tissue Culture treated 6 well plates
Cells: Ficoll Hypaque prepared PBMC

1. Place 10x10^6 cells per well in 1ml of DC Media-use 12 wells if enough cells (2 plates)
2. Place plate at 37C for 2 hours
3. Check plate under microscope to be certain cells have adhered to plate
4. Remove media and nonadhered cells very carefully then gently wash with 2ml of PBS
   • place pipet on side wall of well and don't scrape down the middle of the well
   • save removed media/nonadhered cells just in case cells did not adhere well
5. Check plate to insure presence of adhered cells
6. Add 2 mls of DC Media w/ cytokines to each well
7. Feed with DC Media + cytokines every 2 days
8. At 2 ½ weeks, check cells by flow

Protocol courtesy of Dr. Burlingham's lab