I'm a working on vascular leakage caused by virus infection. For this, I’m trying to standardize the permeability assay with HMEC (5×104 cells/insert) using corning inserts (0.33 mm, 0.4 um) and 40 KD FITC-Dextran, but unfortunately facing a lot of discrepancies. There will be no difference between the treated or infected and even the positive control (TNF). However, I have tried with overnight coating with firbonecting and also 1hr coating as recommended by the manufacturer but could not get consistent results. There is significant variability among the readings of the insert repeats. I tried to use TNF 100 ng/ml as positive control but that also didn’t give any significant difference compared to control at 24 hrs. it will be a great help if anyone can share some suggestions to improve this.
I have also stained the insert to observe the check the monolayer and is appears fine. I have attached pic of the same here.
Thanks in advance Ayan
Hi Ayan,
I use followed protocol for HMEC permeability:
Briefly, HMEC-1 (2 x 10^5) were seeded in 24-well collagen and fibronectin-coated transwells (6.5 nm diameter, 0.4 um pore size, Costar, Corning Inc., Corning, NY) and cultured in the Complete Medium 200 (Cascade Biologics) with low serum growth supplement (LSGS). On day 1 post plating, add your treatment into the upper chamber. HRP (0.5 ug in 10 ul) was added to the upper chamber of transwell for 15 min before harvesting medium from lower chamber (10 ul) to determine HRP activity by incubating with TMB (3,3′,5,5′-tetramethylbenzidine) for 20 min, and analyzed by spectrophotometer at OD450 nm after adding sulfuric acid to stop reaction.
Hope this can help you, good luck!
Andea
Thanks a lot Andea. I'm also trying to do the coating with both collagen and fibronectin. Hope it will work. thanks again Pei-Shan Sung .
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