Can anyone suggest an agent I could use as a positive control for endosome escape? I need something to which to compare my attempts at triggering endosome escape with photothermal heating.
I'm particularly interested in a reagent which may be added to cells just prior to fluorescence imaging and which releases contents from endosomes and lysosomes, ideally without immediately killing the cell. I don't care about long term cell viability or clinical efficacy or anything like that for this control agent. I just need to get a good fluorescent picture of released endosome contents.
Hi Scott,
You may try our endosomal escape technology Photochemical Internalization (PCI). With PCI you can release molecules that are sequestered in endocytic vesicles. Cells are co-incubated with a PCI-photosensitizer (TPCS2a) and a drug/molecule of interest over night. Wash cells with culture medium, chase in drug-free medium for 4 hrs and then light expose the cells with blue light (or red laser with wavelength that match the absorption of the photosensitizer) Note: low fluence rates (10-100 mW/cm2 and low doses (0.5-5 J/cm2). After 30-60 min you perform fluorescence microscopy and check for cytosolic release. The photosensitizer emit red light (>620 nm) when exposed to blue light (400-420 nm). Control cells (without light) will have granular fluorescence (spots) that resemble the shape of endosomes and lysosomes, while light exposed (PCI) cells will have a diffuse fluorescence that is a sign of endosomal escape. You need a photosensitizer, a light source (I guess you have some since you work with phototheraml effects, microscopy light is an alternative) and your molecule of interest (FITC, and Alexa-488 is OK). If you find this attractive, I can give you more details. Check the attached review.
Good luck!
Article Photochemical internalization provides time- and space-contr...
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