Hi, I would like to know how to generate OVA peptide-pulsed dendritic cells in vitro.
Dendritic cell would be co-culture with OT-II T cells, and measured T cell proliferation.
1. How long do I need to incubate DC with peptide? I know two different times, but I don't know what is correct!
1) 37'c, 24h
2) 37'c, 30min
2. Please let me know the good concentration of peptide for OVA peptide-pulsed DC! [ in ug unit ]. In our lab protocol, 10ug/ml concentration would be recommended. But there are no cell number, so I want to know the good cell number and peptide concentration for in vitro work!!
for example) 1x10^6 cells with 0.5ug/ml concentration of peptide
3. 96 well plate is okay for in vitro work?
4. I heard that I have to co-culture the dendritic cell(OVA pulsed) and OT-II and also "additional OVA-peptide". In our lab protocol, 5ug/ml concentration would be recommended, is it okay?
5. I will co-culture the DC and OT-II in 3:1(DC:T) ratio. Is there any recommends?
All the seniors who made stock and protocol are graduated, so I have a hard time.
And there is no one to ask this...
I'm very happy to be able to share your experience!
Thanks for your help!!
Hi....
To generate OVA peptide-pulsed dendritic cells (DCs) in vitro, you can follow the following protocol:
Obtain OVA peptide and DCs. The OVA peptide should be at least 95% pure and the DCs should be purified and healthy.
Cultivate the DCs in a suitable cell culture medium in a tissue culture flask or well plate.
Once the DCs have reached 70-80% confluence, remove the medium and wash the cells with phosphate-buffered saline (PBS).
Add the OVA peptide to the DCs at a concentration of 10-100 µg/ml, depending on the desired level of pulsing. Incubate the cells at 37°C for 24 hours.
After incubation, remove the OVA peptide and wash the DCs with PBS. The OVA peptide-pulsed DCs are now ready for use in your experiment.
It is generally recommended to use a concentration of 10 µg/ml for OVA peptide pulsing. This concentration has been shown to be effective in a number of studies. However, the optimal concentration may vary depending on the specific characteristics of your DCs and the OVA peptide.
A 96 well plate is suitable for in vitro work, but you may want to consider using a larger well plate if you are working with a large number of cells or if you need more space for your experiments.
It is generally recommended to use a 3:1 DC:T cell ratio when co-culturing DCs and T cells. However, the optimal ratio may vary depending on the specific characteristics of your cells and the goals of your experiment.
I hope this information is helpful.
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