3 replies to this topic

[Crimsonbeard]

Hi,

I am trying to label macrophages with nanoparticles and am running into a problem with recovering cells after labeling.  I have tried scraping and trypsin digest up to 6 min, but the cells are still stuck or I rip them apart with the scraping.  

I think an easy way around this problem is to keep  the macrophages from adhering during the 2 hour incubation with the nanoparticles.  Do you know of a way to stop cells from adhering for 2 hours?

Thanks!

[almost a doctor]

I used to grow macrophages in bacterial petri dishes (ie. non-tissue culture treated). Following incubation for up to 7-8 days  I'll treat remove media, wash with warm PBS and then treat with

3 mM EDTA and 10 mM glucose in PBS  for about 5min in the incuabtor to recover the cells.  I never calculated recovery as such, but there were hardly any cells left in the plate.

Hope this helps

Edited by almost a doctor, 27 June 2012 - 08:07 AM.

[dpo]

You can check out the plates from Nunc with UpCell Surface (http://www.nuncbrand...e.aspx?ID=11850). Although I have never used these plates, it may come in handy for your purpose. Apparently, shifting the plates to temperatures lower than 32 °C reduces adhesion to the plates and your cells detach without any other intervention. Of course you have to judge whether this temperature shift is permitted in the assay you are planning.

[rhombus]

Crimsonbeard, on 27 June 2012 - 07:17 AM, said:

Hi,

I am trying to label macrophages with nanoparticles and am running into a problem with recovering cells after labeling.  I have tried scraping and trypsin digest up to 6 min, but the cells are still stuck or I rip them apart with the scraping.  

I think an easy way around this problem is to keep  the macrophages from adhering during the 2 hour incubation with the nanoparticles.  Do you know of a way to stop cells from adhering for 2 hours?

Thanks!

Why not try and label them in SUSPENSION.........no need then to trypsinise

WE use TECHNE biological stirrer bottles for our suspension cultures as they are borosilicate glass and do not allow cells to attach. We grow our 2 x types of murine macrophage (J774 and RAW 264.7) cell lines like this...............  because if you grow them on plastic they are impossible to remove with trypsin

Hope this is useful

Kindest regards

Uncle Rhombus