I labeled my cells with pHrodo Red dextran dye to examine endocytosis of my adherent cells. After labeling with the recommended concentration in PBS, the cells rounded up and dissociated. Is this a problem with this reagent?
pHrodo Red dextran dye is not known to be toxic to cells. What is more likely is that the cells are not healthy in PBS in the amount of time needed for the assay. Instead, we recommend labeling in a more physiological buffer, such as HBSS, or in phenol red-free media, at an optimal temperature for your cells.
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I am performing a phagocytosis assay of macrophages engulfing pHrodo-labeled bacteria. What do you recommend for fixation after the phagocytosis?
pHrodo is relatively non-fluorescent until it enters the acidic phagosome, at which point its fluorescence increases. If you fix the sample, the pHrodo will only reflect the pH of the buffer the cells are in, and not the pH of the phagosome. For this reason, we do not recommend fixing samples. If you want to see how many cells engulfed the labeled bacteria, fix the cells and then place the fixed cells in an acidic buffer for the assay.
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What is the optimal working concentration for pHrodo Red and Green Dextran, 10,000 MW, for Endocytosis (Cat. Nos. P10361, P35368)?
The working concentration for pHrodo Red and Green Dextran, 10,000 MW, for Endocytosis (Cat. Nos. P10361, P35368) is anywhere between 10 and 100 µg/mL, depending on cell type, level of background, duration of loading, and the detection modality. We recommend starting with 50 µg/mL treatment and then titrating up or down to get an optimal signal.
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