Hi! I have cultured human CD34+ cord blood derived CD141+ dendritic cell (DC) using IL-4 and GM-CSF and have achieved around 30% CD141+ DCs. Now, I'm planning to stimulate these DCs with poly I:C (TLR3 agonist) and then proceed with co-culture with CD8+ T-cells. The literature available on the co-culture experiments are confusing to me. Could anybody please share any detailed standard protocol which is either available online or they have experience in DC-T cell co-culture experiments. Any help will be appreciated. Thanks in advance.
Aishwarya Sen,
For DC-CD8+ T cells culture.
1. Ratio matters DC:CD8 ( 1:5 or 1:10), you can try both. (20,000:100,000)
2. Media - RPMI, FCS (10%), PS (1%).
3. Co-culture - 7 days ( change the media on 4th day).
What was the concentration of your GM CSF and IL4 for yours DCs?
For CD8 isolation, which kit did you use? What was your source for CD8 isolation?
Hello, @Paul David. Thanks for the information. I have used 20ng/ml of IL4 and 50ng/ml GM-CSF. I did not use any kit. Instead I sorted the CD11C+HLADR+CD141+CLEC9A+ DCs.
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