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Phagocytosis Detection Kit Red

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RUO
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a. Phagocytosis-Detection-Kit_Live-Cell-Imaging-_082729.
Live-cell fluorescence imaging of RAW264.7 cells treated with with Phagocytosis Detection Beads (red) in complete growth media for 4 hours. Cells were then stained with Viability Dye Green (green) for 30 minutes and nuclei were stained with Hoechst 33342 (blue). Images were captured using a 60X objective. Scale bar: 10 µm
  • a. Phagocytosis-Detection-Kit_Live-Cell-Imaging-_082729.
    Live-cell fluorescence imaging of RAW264.7 cells treated with with Phagocytosis Detection Beads (red) in complete growth media for 4 hours. Cells were then stained with Viability Dye Green (green) for 30 minutes and nuclei were stained with Hoechst 33342 (blue). Images were captured using a 60X objective. Scale bar: 10 µm
  • b. Phagocytosis-Detection-Kit_FC-_082729.
    THP-1 cells were treated with Phagocytosis Detection Beads and Viability Dye Green for 1 hour (filled histogram) or left untreated (open histogram). Cells were analyzed on flow cytometer using PE channel.
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Description

Phagocytosis is a complex process for the ingestion and elimination of pathogens. It is also important for the elimination of apoptotic cells, hence fundamental for tissue homeostasis. Phagocytosis consists of recognition and ingestion of particles larger than 0.5 µm into a plasma membrane-derived vesicle, known as phagosome. Professional phagocytes include monocytes, macrophages, neutrophils, dendritic cells, osteoclasts, and eosinophils. These cells are in charge of eliminating microorganisms and presenting them to cells of the adaptive immune system. In addition, fibroblasts, epithelial cells, and endothelial cells can also perform phagocytosis. These nonprofessional phagocytes cannot ingest microorganisms but are important in eliminating apoptotic bodies. Once internalized, the phagosome fuse with lysosomes to form secondary phagolysosomes for digestion, resulting in progressive decrease of pH. The Phagocytosis Detection Beads are non-fluorescent
outside of cells. However, its fluorescence dramatically increases as they are internalized and within the acidic phagosomes/phagolysosome
compartments.

Product Details
Technical data sheet

Product Details

Verified Reactivity
Human, Mouse
Reported Reactivity
All Species
Formulation
1 vial Phagocytosis Detection Beads, 15 µL
1 vial Viability Dye Green, 20 µL
1 vial DMSO, 100 µL
Storage & Handling
4°C
Application

FC - Quality tested
Live cell imaging - Verified
Recommended Usage

Refer to protocol

Application Notes

Component:
1 vial (15 µL) Phagocytosis Detection Beads (2-8°C, protected from light)
1 vial (20 µL) Viability Dye Green (2-8°C, protected from light)
1 vial (100 µL) DMSO (room temperature)

Required Materials Not Included:
Buffer: Phosphate-buffered saline (PBS), phenol-free media, or HBSS
Complete growth media (cell type dependent)

Detection/Imaging Guidelines:
Phagocytosis Detection Beads: Ex/Em = 570/600 nm
Fluorescence microscope filter set: Texas Red
Flow cytometry channel: PE with yellow/green laser excitation
Viability Dye Green: Ex/Em = 490/525 nm
Fluorescence microscope filter set: FITC
Flow cytometry channel: FITC

Prepare Reagents:

  1. Bring all three components: Phagocytosis Detection Beads, Viability Dye Green, and DMSO. Allow them to reach room temperature.
  2. Prepare 400X stock solution of Viability Dye Green by adding 20 µL of DMSO.
  3. Prepare 12X solution of Viability Dye Green in complete growth media by adding 5 µL of the 400X stock solution of Viability Dye Green to 2 ml growth media. Mix well.
  4. Prepare 12X solution of Phagocytosis Detection Beads in complete growth media by adding 8 µL in 2 ml growth media. Mix well.

Live-Cell Imaging Assay Protocol:

  1. Add the 12X Phagocytosis Detection Beads solution directly into each well of adherent cells growing in complete media. Cells should be about 80% confluent. For example, add 16 µL of 12X Phagocytosis Detection Beads solution to a well containing cells in 200 µL media.
  2. Incubate cells at 37°C for 4 hours.
    Note: Probe concentration and incubation time may need to be adjusted according to cell type and assay conditions.
  3. Add the 12X Viability Dye Green solution to the wells. For example, add 16 µL to 200 µL media.
    Optional: Include live-cell nuclear counterstain such as DRAQ5™ (Cat. No. 424101).
  4. Incubate cells at 37°C for 30 minutes.
  5. Gently wash cells twice with PBS, phenol-free media or HBSS.
  6. Add sufficient amount of PBS, phenol-free media, or HBSS to the wells and proceed immediately to live-cell imaging.
Flow Cytometry Assay Protocol:
  1. Resuspend cells (1-2 x 106) in 200 µL of PBS, HBSS, or Cell Staining Buffer (Cat. No. 420201).
  2. Add 16 µLof 12X Phagocytosis Detection Beads working solution
    Note: Prepare single color tubes for cell only, PE, and FITC for compensation.
  3. Add 16 µL of 12X Viability Dye Green staining solution to the cells.
  4. Incubate at 37°C for 1 hour.
  5. Wash cells twice with PBS, HBSS or Cell Staining Buffer
  6. Resuspend in 200-300 µL of PBS, HBSS, or Cell Staining Buffer
  7. Proceed to flow cytometry analysis using an appropriate filter set.

Antigen Details

Distribution

Phagocytes

Function
Phagocytosis activity
Cell Type
Dendritic cells, Macrophages
Biology Area
Cell Biology, Immunology
Molecular Family
Organelle Markers
Gene ID
NA
Go To Top Version: 1    Revision Date: 08/27/2024

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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