In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles® indicators—bacteria and yeast labeled with fluorescent dyes.

Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used monitor stages in the pathway.

 

 

No-wash assays

A very fast and highly accurate way to monitor stages in the phagocytosis pathway uses pHrodo™ indicators. Both pHrodo™ Red and pHrodo™ Green are conjugated to a range of particles for phagocytosis measurement with no quench or wash required.

pHrodo™ dyes are essentially non-fluorescent at neutral pH and exhibit increasing signal with a red or green readout respectively as the pH decreases. The increase in fluorescent signal can be used to monitor progression in the phagocytic pathway.

phagocytosis

Mouse monocyte/macrophage cells (MMM cells, ATCC) labeled with NucBlue® Live ReadyProbes® Reagent and incubated for 60 minutes with pHrodo™ Red E. coli in Live Cell Imaging Solution.

Quench or wash-based assays

Quench or wash-based assays are useful to indicate phagocytosis with uniform signal that is often used as a control for phagocytosis measurement.

Alexa Fluor® dyes conjugated to a range of particles provide a choice of wavelength options and single-emission measurement for each. Fluorescein provides a pH-sensitive signal that decreases with acidification of the phagosome.

phagocytosis

MMM macrophage cells incubated with Zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 594 Conjugate and washed in Live Cell Imaging Solution before imaging.

Phagocytosis selection guides

 
Readout
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
Range
Monitors early phagosome to early lysosome formation
Vehicle
S. aureus
E. coli
Zymosan A
Zymosan A
E. coli
S. aureus
Common filter set
TRITC
FITC
Labels
pHrodo™ Red
pHrodo™ Green
Ex/Em (nm)
500/585
509/533
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
I, M, FC
Formats*
5 x 2 mg
5 x 2 mg
5 x 1 mg
5 x 1 mg
5 x 2 mg
5 x 2 mg
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.
 
Readout
Measures phagocytic activity in whole blood samples by flow cytometry
Range
 Monitors phagosome formation
Vehicle or Method
E. coli
Label your own particles
E. coli 
S. aureus 
Common filter set
TRITC
FITC
Labels
pHrodo™ Red
pHrodo™ Green
Ex/Em (nm)
500/585
509/533
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
Flow cytometry 
Format
1 kit
1 kit
1 kit
1 kit
Cat. No.
 
Readout
Requires quenching or washing; internalized dye fluoresces, external dye is quenched/washed 
Range
Stable signal with no modulation throughout the phagocytic process 
Vehicle or Method
S. aureus
E. coli
Zymosan A
 Zymosan A
E. coli
S. aureus
Common filter set
FITC
Texas Red®
Labels
Alexa Fluor® 488
Alexa Fluor® 594
Ex/Em (nm)
494/517
590/617
Signal-to-noise ratio
Photostability
Fixability
Image cells live or after fixing
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
Imaging 
Format
2 mg
2 mg
2 mg
2 mg
2 mg
2 mg
Cat. No.
 
Readout
Internalized dye fluoresces, requires negative control subtraction
Range
Fluorescence decrease in early phagocytosis 
Vehicle
E. coli
E. coli
S. aureus
Zymosan A
Common filter set
FITC
Labels
Fluorescein
Ex/Em (nm)
480/520
Signal-to-noise ratio
Photostability
Fixability
Image cells live or after fixing
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms*
I, M
Imaging
Format
1 kit
10 mg
10 mg
10 mg
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.
No-wash assays
 
Readout
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
Range
Monitors early phagosome to early lysosome formation
Vehicle
S. aureus
E. coli
Zymosan A
Zymosan A
E. coli
S. aureus
Common filter set
TRITC
FITC
Labels
pHrodo™ Red
pHrodo™ Green
Ex/Em (nm)
500/585
509/533
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
I, M, FC
Formats*
5 x 2 mg
5 x 2 mg
5 x 1 mg
5 x 1 mg
5 x 2 mg
5 x 2 mg
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.
No-wash, whole blood
 
Readout
Measures phagocytic activity in whole blood samples by flow cytometry
Range
 Monitors phagosome formation
Vehicle or Method
E. coli
Label your own particles
E. coli 
S. aureus 
Common filter set
TRITC
FITC
Labels
pHrodo™ Red
pHrodo™ Green
Ex/Em (nm)
500/585
509/533
Signal-to-noise ratio
Photostability
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
Flow cytometry 
Format
1 kit
1 kit
1 kit
1 kit
Cat. No.
Wash-based assays, stable signal
 
Readout
Requires quenching or washing; internalized dye fluoresces, external dye is quenched/washed 
Range
Stable signal with no modulation throughout the phagocytic process 
Vehicle or Method
S. aureus
E. coli
Zymosan A
 Zymosan A
E. coli
S. aureus
Common filter set
FITC
Texas Red®
Labels
Alexa Fluor® 488
Alexa Fluor® 594
Ex/Em (nm)
494/517
590/617
Signal-to-noise ratio
Photostability
Fixability
Image cells live or after fixing
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms
Imaging 
Format
2 mg
2 mg
2 mg
2 mg
2 mg
2 mg
Cat. No.
Wash-based, decreasing signal
 
Readout
Internalized dye fluoresces, requires negative control subtraction
Range
Fluorescence decrease in early phagocytosis 
Vehicle
E. coli
E. coli
S. aureus
Zymosan A
Common filter set
FITC
Labels
Fluorescein
Ex/Em (nm)
480/520
Signal-to-noise ratio
Photostability
Fixability
Image cells live or after fixing
Bibliography
Multiplexing
Yes
Live cells
Yes
Fixed cells
No
Fixable
Yes
Platforms*
I, M
Imaging
Format
1 kit
10 mg
10 mg
10 mg
Cat. No.
* I = Imaging, M = Microplate, FC = Flow cytometry.

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