I am trying to optimize an in vitro BBB model to study lymphocyte migration from the blood stream into the CNS.
In my set up I use a 24-well plate with Transwell inserts of 8µm pore-size. The inserts are coated with Gelatin 0,1% and bEnd.3 endothelial cells are grown on top until confluent.
Then I add EL-4 cells in suspension in the upper chamber, and media with a chemokine (CCL-19 20µg/ml) in the lower chamber, and I let them migrate for 4 hours.
I am currently counting the migrated EL-4 cells (in the lower chamber) manually, but this gives me a lot of variation. Since there are so few cells, I spin them down to try to concentrate them, and I think I might lose many if not all cells in that step.
Have anyone tried with radioactive lavelling, RNA quantification or some other method?
Thanks in advance,
Alba Manresa
Take a look to this method. High sensitivity.
https://www.researchgate.net/publication/266209989_Quantitative_method_for_in_vitro_matrigel_invasiveness_measurement_through_image_analysis_software
Article Quantitative method for in vitro matrigel invasiveness measu...
Thank you very much for your suggestion Dr. Vamsi krishna Karuturi.
I have also been recommended to use the Accuri flow cytometer, so I will give this a try. But I will definetely give a thought to your suggestion as well, thanks again!
Dear Javier Dotor, thanks a lot for your answer.
I am affraid I cannot use your matrigel system, as lymphocytes don't migrate through matrigel -at least in my hands. I think they need the interactions with the receptors of the endothelial cell layer to migrate.
Therefore, the cells I want to quatify are in suspension in the lower chamber, not embedded in the matrigel, so I don't think I can apply this imaging system.
But I could use it in other experiments, so thank you for your input, very helpful!
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