Hello,
I would like to use s. aureus enterotoxin B (SEB) for conjugate formation of T cells and B cells (live microscopy). I want to use SEB because it is available from SIGMA, whereas SEE is more difficult to obtain.
What is the difference between SEE and SEB in terms of conjugate formation?
Can I use SEB pulsed Rajis for conjugate formation with primary CD4+ human/mice and Jurkat T cells? I heared that SEB is not suitable for Jurkats, do you have experience on this matter? I would also be happy about detailed protocols.
Thanks for helping,
Niko
Hi Nikolaos
Thanks for query
But, I don't have experience on this.
Sorry of that.
Rajni Kant
Dear Nikolaos Tsopoulidis
Pls. find the attached files,
Hoping this will be helpful to your project, good luck
Regards
Prof. Houda Kawas
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2605221/
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156429
http://www.nature.com/cdd/journal/v22/n10/fig_tab/cdd201517f4.html
http://jcb.rupress.org/content/209/1/143
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2528574/
Also
Good luck
http://www.bio-protocol.org/e1758
http://jcb.rupress.org/content/198/6/1025
http://www.jimmunol.org/content/160/5/2072
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163173/
thank you very much. So the conclusion is SEB for primary T cells SEE for primary and Jurkat T cells
I have established now the protocol for primary CD4+ T cells and Raji B cells:
rpmi++=medium plus antibiotics and 10% serum
Format: angiogenesis slides for live cell imaging coated with poly-lysine
T cells: primary human CD4+ T cells transduced for lifeact expression
-On the day of imaging: Incubate Raji cells (1 × 1E6 cells/ml) in 1:1000 (10 μM) CMAC and
1:200 (5µg/ml) SEB in serum free media not PBS for 30 min at 37°C
-Wash Raji cells with RPMI++ , and resuspend in RPMI++
-Adjust T cells to 0.5E5/20µl (2.5E6/ml)
Prepheat microscope environmental control chamber, humidify and gas with CO2
-B cell to T cell ratio should be 1:1, Note: T cells engaging in multiple synapses show reduced
F-actin at each synapse also transfer cells shortly before imaging from cell culture -Add into one well of an angiogenesis slide 0.5E5 T cells (in 20 µl primary cell medium) and
wait 5-10 min for the cells to adhere to the poly-lysine coated surface
-Add 20 µl containing 5E4 SEB loaded CMAC stained Raji B cells DROPWISE -Start imaging and acquire min. 30 min sequence
Hi Nikolaos,
I am going to start to use Jurkat cells and SEB/SEE-pulsed Raji B cells for live microscopy. Did you finally use SEE for Jurkat cells? Is the protocol similar to the one that you described for CD4+ T cells?
Thanks,
Blanca
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