Phagocytosis
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.
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.
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. | ||||
| 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. | ||||