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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Can I store reconstituted pHrodo BioParticles Conjugates for Phagocytosis and Phagocytosis Kit, for Flow Cytometry?
Yes. Once reconstituted, pHrodo BioParticles Conjugates for Phagocytosis and Phagocytosis Kit, for Flow Cytometry (Cat. Nos. P35367, P35361, P35360, P35366, P35364, P35365, A10010) can be stored at 2 - 8 degrees C for several weeks, as long as sodium azide is added to a final concentration of 2 mM. If no sodium azide is added, the cell suspension needs to be used right away or on the same day to avoid contamination. DO NOT FREEZE the resuspended pHrodo bioparticle conjugates.
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Are the Invitrogen BioParticles products sterile?
While the bacteria have been attenuated with formaldehyde and alcohol desiccation, the BioParticles products are not considered sterile, and we do not recommend incubation of more than 4 hours. This applies to all of our dye-labeled (pHrodo, Alexa Fluor, etc.) and unlabeled BioParticles products.
What is the concentration of bacterial particles for pHrodo BioParticles Conjugates for Phagocytosis?
The bacterial particles per weight for pHrodo BioParticles Conjugates for Phagocytosis, is as follows:
pHrodo E. Coli BioParticles Conjugate for Phagocytosis (Cat. Nos. P35360, P35361, A10025) contains 3 x 108 particles/mg.
pHrodo S. aureus BioParticles Conjugate for Phagocytosis (Cat. Nos. A10010, P35367) contains 3 x 108 particles/mg.
pHrodo Zymosan BioParticles Conjugate for Phagocytosis (Cat. No. P35365) contains 2 x 107 particles/mg.
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What is the type of bond that attaches the dyes to the BioParticles probes?
We use amine-reactive dyes to covalently attach fluorescent dyes to all of our BioParticles probes such as the Escherichia coli (K-12 strain) BioParticles probes, Staphylococcus aureus (Wood strain without protein A) BioParticles, and the Zymosan A (S. cerevisiae) BioParticles probes.
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