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. pHrodo Deep Red, Red, and 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.
MMM macrophage cells were incubated with pHrodo Deep Red E. coli BioParticles for 90 minutes then stained with NucBlue Live and Calcein 45 minutes prior to image acquisition.
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.
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
pHrodo Green
E. coli BioParticles Conjugate for Phagocytosis
|
pHrodo Red
E. coli BioParticles Conjugate for Phagocytosis
|
pHrodo Deep Red
E. coli BioParticles Conjugate for Phagocytosis
| ||
|---|---|---|---|---|
| Readout |
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
| |||
| Range |
Monitors phagosome formation
| |||
| Vehicle | E. coli | |||
| Common filter set |
FITC/GFP
|
TRITC/RFP
|
Cy5
| |
| Label |
pHrodo Green
|
pHrodo Red
|
pHrodo Deep Red
| |
| Ex/Em |
509/533
|
560/585
|
640/655
| |
| Signal-to-noise ratio |  |  |  | |
| Photostability | | | | |
| Brightness | | | | |
| Bibliography | ||||
| Multiplexing |
Yes
| |||
| Live cells |
Yes
| |||
| Fixed cells |
No
| |||
| Fixable |
Yes
| |||
| Platforms |
Imaging, microplates, flow cytometry
| |||
| Format |
5 x 2 mg
|
5 x 2 mg
|
5 x 2 mg
| |
| Cat. No. | ||||
pHrodo Green
S. aureus Bioparticles Conjugate for Phagocytosis
|
pHrodo Green Zymosan Bioparticles Conjugate for Phagocytosis
|
pHrodo Red
S. aureus Bioparticles Conjugate for Phagocytosis
|
pHrodo Red Zymosan Bioparticles Conjugate for Phagocytosis
| |
|---|---|---|---|---|
| Readout |
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
| |||
| Range |
Monitors phagosome formation
| |||
| Vehicle | S. aureus |
Zymosan A
| S. aureus |
Zymosan A
|
| Common filter set |
FITC/GFP
|
TRITC/RFP
| ||
| Labels |
pHrodo Green
|
pHrodo Red
| ||
| Ex/Em (nm) |
509/533
|
560/585
| ||
| Signal-to-noise ratio | | | ||
| Photostability | | | ||
| Brightness | | | ||
| Bibliography | ||||
| Multiplexing |
Yes
| |||
| Live cells |
Yes
| |||
| Fixed cells |
No
| |||
| Fixable |
Yes
| |||
| Platforms |
Imaging, microplates, flow cytometry
| |||
| Formats* |
5 x 2 mg
|
5 x 1 mg
|
5 x 2 mg
|
5 x 1 mg
|
| Cat. No. | ||||
| 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) |
560/585
|
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. | ||||
pHrodo Green
E. coli BioParticles Conjugate for Phagocytosis
|
pHrodo Red
E. coli BioParticles Conjugate for Phagocytosis
|
pHrodo Deep Red
E. coli BioParticles Conjugate for Phagocytosis
| ||
|---|---|---|---|---|
| Readout |
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
| |||
| Range |
Monitors phagosome formation
| |||
| Vehicle | E. coli | |||
| Common filter set |
FITC/GFP
|
TRITC/RFP
|
Cy5
| |
| Label |
pHrodo Green
|
pHrodo Red
|
pHrodo Deep Red
| |
| Ex/Em |
509/533
|
560/585
|
640/655
| |
| Signal-to-noise ratio | | | | |
| Photostability | | | | |
| Brightness | | | | |
| Bibliography | ||||
| Multiplexing |
Yes
| |||
| Live cells |
Yes
| |||
| Fixed cells |
No
| |||
| Fixable |
Yes
| |||
| Platforms |
Imaging, microplates, flow cytometry
| |||
| Format |
5 x 2 mg
|
5 x 2 mg
|
5 x 2 mg
| |
| Cat. No. | ||||
pHrodo Green
S. aureus Bioparticles Conjugate for Phagocytosis
|
pHrodo Green Zymosan Bioparticles Conjugate for Phagocytosis
|
pHrodo Red
S. aureus Bioparticles Conjugate for Phagocytosis
|
pHrodo Red Zymosan Bioparticles Conjugate for Phagocytosis
| |
|---|---|---|---|---|
| Readout |
No-wash, no-quenching fluorescence intensity assay format, where fluorescence increases throughout phagocytic process
| |||
| Range |
Monitors phagosome formation
| |||
| Vehicle | S. aureus |
Zymosan A
| S. aureus |
Zymosan A
|
| Common filter set |
FITC/GFP
|
TRITC/RFP
| ||
| Labels |
pHrodo Green
|
pHrodo Red
| ||
| Ex/Em (nm) |
509/533
|
560/585
| ||
| Signal-to-noise ratio | | | ||
| Photostability | | | ||
| Brightness | | | ||
| Bibliography | ||||
| Multiplexing |
Yes
| |||
| Live cells |
Yes
| |||
| Fixed cells |
No
| |||
| Fixable |
Yes
| |||
| Platforms |
Imaging, microplates, flow cytometry
| |||
| Formats* |
5 x 2 mg
|
5 x 1 mg
|
5 x 2 mg
|
5 x 1 mg
|
| Cat. No. | ||||
| 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) |
560/585
|
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. | ||||
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