Hello,
we've been running Amyloid Beta uptake assays in cultured astrocytes, where at the end we measure several parameters which include media Ab presence, cell lysate and ICC for Ab.
We follow the following protocol, published by Lin et al 2018:
To prepare oligomerized Ab42,Ab42 peptide (AnaSpec) was dissolved in 1%NH4OH at 1 mg/ml and sonicated. Lyophilized Ab42 was further dissolved in water, filtered and incubated at 37C for 1 day before use. iPSC-derived astrocytes were seeded in 24-well plates (3X10^4 /well) for 2 days and incubated with oligomeric Ab42 (we performed dosage curve of 0, 62.5, 125, 187.5, 250 and 375ng/ml) for additional 2 days. Ab42 oligomer was also added to media only (without cells) to measure total levels of Ab42 2 days after culture.
The major problem is trying to quantify Ab uptake by ICC. As we increase the dosage, we can see a nice uptake in Ab in the cells, but we also get blotches and tiny stringy artifacts which do not seem cell associated. As if they aggregated and got stuck on the cells or on the plate matrix.
We perform several washes with X1PBS before fixing the cells for ICC and we cant get rid of those artifacts.
The problem with this is we cannot relay on quantification by imaging as the flouresence they add is very high, and their aggregation also changes the available Ab that needs to be uptaken by the astrocytes, thus changing the intended concentration of the Ab.
Any solution for this problem will be highly appreciated.
Oded