Does anyone know how many mouse spleen cells should be stimulated with how much Concanavalin A? How for long to see a considerable number of spots?
Hi Puja,
In the rat spleen cell assay I used to run, we would stimulate for 48 hrs using 5micrograms/ml Con A. Clumping usually was visible after about a day or so. To measure the proliferation, we would then use a MTT assay.
Thanks, Daniel,
Can you also suggest no. of cells and amount of Concanavalin ?
Puja
Hi Puja,
We would use 1 x 10^5 cells per ml, with 250ul per well in a 96 well plate. Generally we would use Con A at a final well concentration of 5ug/ml. This was achieved by adding 50ul of the culture media (in our case, RPMI 1640, 2mM L-Glutamine, 10% FBS) with 30ug/ml Con A to each well. From what I've read you may be able to do it with half this concentration of Con A, but the above was the established protocol in our lab.
Depending on what assay you are using to measure the proliferation (MTT, Radiolabeling etc) you may need to adjust the cell number to get an appropriate signal.
Thanks a lot! Daniel
One last question are you using Concanavalin from Sigma if not then from where and the cat no.
An additional remark: for you initial acivation tests you might want to compare ConA resolved in Ringer solution or PBS, respectively, since they differ both in presence/absence (and complexation) of calcium ions and pH with ConA beeing a calcium binder and changing its structure with pH.
One might think that this is not important since the ConA solution is added to a higher amount of culture medium anyhow, but to my knowledge even in in vivo models of ConA-mediated liver injury, some groups which dissolved their compounds in PBS by default, had problems to have a pronounced effect, whereas others who dissolved ConA in Ringer solution had not.
However, I'm not aware of any back-to-back comparison...just a suggestion.
It's important to titrate the ConA, since different batches differ in efficacy, and the window of effective concentration can be relatively small. Usually btw 2-10 ug/ml works well. If you're measuring proliferation, it's important to look at the wells visually to confirm that stimulation has not exhausted the medium prior to sample collection (medium turns yellow), as this will give incorrectly low readouts. Also, the batch of serum can strongly influence the magnitude of the response, as can adding a reducing agent to the medium.
We used 1 million splenocytes (mice) in 6-well plate, 5 and 10 ug/ml of Concanavalin-A (Sigma). An increase in cell proliferation (or no of spots as u say..) can be observed starting 24 h of incubation, best observed at 72h. Cytokines can also be studied well from this set up of cell no and conA concentration.
Hi, in mine experience you can use 5ug/ml of ConA for 72-96h and Works perfectly for INFg ELISPOT. For measurement proliferation 3 or 600 000 cell/well could be enough and BrdU stain it's really sensitive.
Good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology