I have been trying to induce inflammation in HUVEC cells using TNF-alpha (5ng/mL) for 4 hrs. However, as the passage number increases the induction is not so strong. I check using ICAM-1 and VCAM-1 antibodies. Do you think as cells go through each passages they lose some TNF receptors?
Hi Snigdha,
Several possibilities spring to mind:
The amount of ICAM-1 and VCAM-1 receptors that the HUVECs can express can depend on its 'growth phase'. Upon seeding the HUVECs try to cover the plate as fast as possible and this can result in a temporary decrease in the amount of adhesion molecules on the cells.
Try to do your tests a couple of days later, when the HUVEC layer is better developed.
Try changing the growth medium. Maybe the cells are fully activated already.
ICAM-1 and VCAM-1 are fairly slowly expressed, as compared to e.g. Selectins. Check the expression after a longer period of TNF exposure.
Are you sure you still have HUVECs? I have seen cultures where HUVECs were slowly 'taken over' by other cells like fibroblasts or where they seemed to de-differentiate (and losing HUVEC-capabilities).
Hope you find the problem.
Best regards,
Ype
Hello Ype,
Thank you so much for you answer. I have tried growing the cells for a longer period, but sometimes I find ICAM-1 and VCAM-1 expression in the untreated sample as well, where I have not added TNF. This is pretty unusual. I do not think there is any contamination also.
Snigdha
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