I would like to evaluate the in vitro uptake of chitosan based nanoparticles by the macrophage cell line Raw 264.7 by flow cytometry. I already performed some confocal uptake studies and I saw that even after wash the cells 6 times with PBS 7.4 at 37ºC there are some particles outside the cells that I cannot remove. I have my chitosan labelled with FITC and I would like to quantify by flow cytometry the amount of cells that will have taken up my nanoparticles in order to compare 2 different chitosan based formulations. But how can I do that if I can't completely remove the remaining nanoparticles of outside the cells? Does anyone know how I can overcome this issue? Is there any solvent or buffer that I could use to wash or destroy my nanoparticles without affecting the cells?
I really appreciate your help.
Thank you.
Dulce
I am not an expert in nanoparticles. But since you are interested in how many nanoparticales enter the cell; after loading the cells with labelled nanoparticles, could you not treat the cells with a light concentration of trypsin to digest off the sticky nanoparticles. You could then check this by microscopy to see if they are still there. If they are binding to connective on the cell surface, slightly digesting it could help.
Hi, I think it could be quite difficult to remove nanoparticles from the cell membrane directly after loading. But if you can waiting for few hours most of nanoparticles will be internalized into perinuclear space. According our observation more than 95%. It also will be quite useful to control SSC to be sure that process is completed.
FCM (Flow cytometry) is among the most popular techniques used to assess nanoparticle uptake. no need to remove nanoparttilces from surface for FCM. Please follow article:
PubMed PMID: 19661298.
Yellepeddi VK, Kumar A, Palakurthi S. Biotinylated poly(amido)amine (PAMAM)
dendrimers as carriers for drug delivery to ovarian cancer cells in vitro.
Anticancer Res. 2009 Aug;29(8):2933-43.
Thank you all for your helpful answers.
I probably will try the approach suggested by Shann Yu, I will treat my cell suspensions with trypan blue. This quenching method was already successfully applied for evaluate the uptake of FITC labelled chitosan particles by confocal microscopy.
you cannot avoid adsorption of nanoparticles on cell surface. there are methods to get rid of that for the evaluation by FACS.
Yes, I cannot avoid adsorption of nanoparticles on cell surface. But I can probably perform a binding assay at 4ºC, at this temperature the uptake is inhibited which allows us to assess only the binding of the particles to cell surface. However, I think that the quenching method with trypan blue will be a better approach for me because this way I can eliminate the interference not only of the nanoparticles adsorbed to the cell surface but also any nanoparticles in suspension that hadn´t been effectively eliminated by washings.
I'm sorry Ajay Kumar but I can't find in your paper the methods that could help me to avoid the interference of the nanoparticles adsorbed at the cell surface. Are you talking about inhibit the endocytosis as you did to assess to uptake mechanism of the dendrimers? Or do you use any computational method? Thank you.
we kept, what you've mentioned above, binding assay at 4ºC as a control for FACS. Cells were washed several times (3X) in PBS (pH 7.4). Extra volume for washing helps in diluting nanoparticles in suspension and it also reduces non-specific interactions of nanoparticles with the cells. non-adsorbed nanoparticles will not be counted by FACS and will be separated with cells debris or waste by FSC/SSC gating. I hope it helps. Endocytosis experiment was conducted to assess targetability of ligand attached onto the surface, which may not be necessary in your case..
Shann Yu's suggestion is very interesting. Do you have a published reference or an example as to how the results would look by flow cytometry. MY e-mail is [email protected]. Would be very interested to learn more.
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