Phagocytosis Assay Kit (IgG FITC)

Phagocytosis Assay Kit (IgG FITC) Item No. 500290

Customer Service 800.364.9897 * Technical Support 888.526.5351 www.caymanchem.com

TABLE OF CONTENTS GENERAL INFORMATION

3 Materials Supplied 4 Precautions 4 If You Have Problems 4 Storage and Stability 4 Materials Needed but Not Supplied

INTRODUCTION 5 Background 5 About This Assay PRE-ASSAY PREPARATION

6 Reagent Preparation

7 Flow Cytometry

ASSAY PROTOCOL

8 Fluorescence Microscopy 9 Plate Reader P ERFORMANCE CHARACTERISTICS 10 Cell Staining 11 Flow Cytometer

GENERAL INFORMATION Materials Supplied Item Number 400291 10009322 400292

Item

Quantity/Size

Storage

Latex Beads-Rabbit IgG-FITC Complex

1 vial/150 µl

4°C

Cell-Based Assay Buffer Tablet

2 tablets

Room Temperature

Trypan Blue (10X)

1 vial/500 µl

Room Temperature

If any of the items listed above are damaged or missing, please contact our Customer Service department at (800) 364-9897 or (734) 971-3335. We cannot accept any returns without prior authorization.

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WARNING: This product is for laboratory research use only: not for

administration to humans. Not for human or veterinary diagnostic or therapeutic use.

RESOURCES 12 Troubleshooting 13 References 13 Related Products 14 Warranty and Limitation of Remedy 15 Plate Template 16 Notes

GENERAL INFORMATION

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Precautions Please read these instructions carefully before beginning this assay. For research use only. Not for human or diagnostic use.

If You Have Problems Technical Service Contact Information Phone: 888-526-5351 (USA and Canada only) or 734-975-3888 Fax: 734-971-3641 Email: [email protected] Hours: M-F 8:00 AM to 5:30 PM EST In order for our staff to assist you quickly and efficiently, please be ready to supply the lot number of the kit (found on the outside of the box).

Storage and Stability This kit will perform as specified if stored as directed and used before the expiration date indicated on the outside of the box.

Materials Needed But Not Supplied 1. A 6-, 12-, 24-, or 96-well plate for culturing cells 2. THP-1, or Raw 264.7 cells (can be obtained from ATCC); other cell lines or blood cells can also be used 3. A fluorescence microscope equipped with a filter capable of measuring excitation and emission at 485 nm and 535 nm, respectively, or a flow cytometer equipped with laser capable of excitation at 488 nm 4. Adjustable pipettes and a repeating pipettor

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GENERAL INFORMATION

INTRODUCTION Background Phagocytosis is a very important physiological process which is characterized by the ingestion of foreign particles and killing of microorganisms by phagocytic leukocytes (granulocytes, monocytes, and macrophages). It provides a first line of host defense against potential pathogens. Phagocytosis is used as a model of microbe-innate immune interactions, resulting in increased understanding of the consequences of these interactions.1 Phagocytosis involves a complex series of events including cytoskeletal rearrangement, alterations in membrane trafficking, activation of microbial killing mechanisms, production of pro- and anti-inflammatory cytokines and chemokines, activation of apoptosis, and production of molecules required for efficient antigen presentation to the adaptive immune system. Aberrant phagocytosis has been implicated in several pathological conditions, such as Multiple Sclerosis, Alzheimer’s disease, and atherosclerosis. Deficiencies in phagocytosis cause severe and recurrent bacterial and fungal infections in affected individuals.2 There are a variety of assays available to study the regulation and mechanism of phagocytosis in vitro. Over time, these assays have changed in nature. There were assays employing microscopy to directly visualize engulfed particles, spectrophotometric evaluation of phagocytized paraffin droplets containing dye, or scintillation counting of radiolabeled bacteria. More recently developed assays use fluorescently-opsonized latex beads or fluorescently-labeled bacteria followed by flow cytometric analysis to assess the degree of phagocytosis. The flow cytometric assay has an advantage of rapid analysis of thousands of cells and quantification of internalized particle density for each analyzed cell. Thus, the degree of phagocytosis from cells treated with different compounds can be compared quantifiably.

About This Assay Cayman’s Phagocytosis Assay Kit (IgG FITC) employs latex beads coated with fluorescentlylabeled rabbit-IgG as a probe for the identification of factors regulating the phagocytic process in vitro. The engulfed fluorescent-beads can be detected using a microscope capable of measuring excitation and emission at 485 and 535 nm, respectively, or be analyzed by a flow cytometer capable of excitation at 488 nm.

INTRODUCTION

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ASSAY PROTOCOL

PRE-ASSAY PREPARATION NOTE: The Latex Bead-Rabbit IgG-FITC Complex is light sensitive. Do not expose to direct intense light.

Reagent Preparation 1. Assay Buffer Preparation Dissolve each Cell-Based Assay Buffer Tablet (Item No. 10009322) in 100 ml of distilled water. This buffer should be stable for approximately one year at room temperature. 2. Latex Beads-Rabbit IgG-FITC Solution Preparation Prepare a Latex Beads-Rabbit IgG-FITC Solution by diluting the bead mixture (Item No. 400291) 1:10 in the culture medium you are using for your cells. Mix well to make sure there are no particles or flakes in the solution. 3. Trypan Blue Quenching Solution Preparation Prepare a Trypan Blue Quenching Solution by diluting the Trypan Blue stock solution (Item No. 400292) 1:10 in the Assay Buffer. Mix well to make sure there are no particles or flakes in the solution.

Flow Cytometry 1. Culture cells in 6-, 12-, or 24-well plates at a density of 5 x 105 cells/ml in a CO2 incubator overnight at 37°C. 2. The next day, treat the cells with experimental compounds or vehicle (each sample should be run in duplicate or triplicate). Add 100 μl of the Latex Beads-Rabbit IgGFITC Solution (see page 6) per ml of culture medium to each well of the plate. For example, if you culture cells in 2 ml of culture medium in a 6-well plate, add 200 μl of the Latex beads-Rabbit IgG-FITC Solution to each well. Mix gently. Further dilution, such as adding only 50 μl of the Latex Beads-Rabbit IgG-FITC Solution to 1 ml of culture medium, may be used depending on the cell type. 3.

Incubate the cells in a CO2 incubator at 37°C for 24-48 hours, or for a period of time according to your normal protocol.

4. Harvest cells from each well into a plastic tube fitted for the flow cytometer. 5. Centrifuge the samples for five minutes at 400 x g at room temperature. Carefully aspirate the supernatant. Add 1 ml of Assay Buffer to each tube and votex to ensure that all cells are suspended. 6. Centrifuge the samples for five minutes at 400 x g at room temperature. Carefully aspirate the supernatant. 7. Repeat steps 5-6 one more time. 8. Add 500 μl of Assay Buffer to each tube and vortex to ensure that all cells are suspended in the assay solution. 9. Analyze the samples immediately in the FL1 channel of a flow cytometer.

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PRE-ASSAY PREPARATION

ASSAY PROTOCOL

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Fluorescence Microscopy

Plate Reader

A 6-, 12-, 24-, or 96-well plate can be used for this method. We recommend that the cell density be less than 1 x 106 cells/ml. Optimal conditions will be dependent on the cell type.

A 96-well black culture plate should be used for this method. We recommend that the cell density be less than 1 x 106 cells/well. Optimal conditions will be dependent on the cell type.

1.

Culture cells in 6-, 12-, 24-, or 96-well plates at a density of 5 x 105 cells/ml in a CO2 incubator overnight at 37°C.

2. The next day, treat the cells with experimental compounds or vehicle (each sample should be run in duplicate or triplicate). In the meantime, add 100 μl of the Latex Beads-Rabbit IgG-FITC Solution (see page 6) per ml of culture medium to each well of the plate. For example, if you culture cells in 2 ml of culture medium in a 6-well plate, add 200 μl of the Latex Beads-Rabbit IgG-FITC Solution into each well. Mix gently. Further dilution, such as adding only 50 μl of the Latex Beads-Rabbit IgGFITC Solution to 1 ml of culture medium, may be used depending on the cell type. 3.


4. The cells can be analyzed directly in the culture medium since phenol red does not interfere with fluorescent staining. Alternatively, if the cells in your experiment have a tendency to attract the Latex Beads-rabbit IgG-FITC complex to the membrane, go to the following steps. The following steps are optional:

1.

Culture cells in a 96-well black plate at a density of 5 x 104 cells/well in 100 μl culture medium in a CO2 incubator overnight at 37°C.

2. The next day, treat the cells with experimental compounds or vehicle (each sample should be run in duplicate or triplicate). In the meantime, add 10 μl of the Latex Beads-Rabbit IgG-FITC Solution (see page 6) to each well of the plate. Mix gently. 3.


4. Centrifuge the plate for five minutes at 400 x g at room temperature. Discard the supernatant by careful aspiration. 5. Add 50 μl of Trypan Blue Solution (see page 6) to each well. Incubate for one to two minutes at room temperature. 6. Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully aspirate the excess Trypan Blue. 7.

Read the fluorescent intensity of each sample in the fluorescence plate reader using an excitation of 485 nm and an emission of 535 nm.

5. Centrifuge the plate for five minutes at 400 x g at room temperature. Discard the supernatant by careful aspiration. 6.

Add 500 μl, 250 μl, 125 μl, or 50 μl of Trypan Blue Solution (see page 6) to each well of 6-, 12-, 24-, or 96-well plate respectively. Incubate for one to two minutes at room temperature.

7.

Centrifuge the plate for five minutes at 400 x g at room temperature. Carefully aspirate excess Trypan Blue. Analyze each sample using a fluorescence microscope equipped with filters for detecting excitation and emission at 485 and 535 nm, respectively.

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ASSAY PROTOCOL

ASSAY PROTOCOL

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PERFORMANCE CHARACTERISTICS

Flow Cytometry

Cell Staining An example of typical staining of phagocytosis obtained using this kit is shown in the figure below. Your results may vary based on the number of cells plated and the cell type you use.

A

B

C

PMA induces the differentiation of THP-1 cells into phagocytic cells, measured by fluorescence Figure 1. THP-1 PMA cells induces the differentiation cells into phagocytic cells, microscopy. were seeded in a 6-well plate atof 5 THP-1 x 105 cells/well and incubated overnight in a measured by fluorescence microscopy. cellsvehicle were(Panel seeded in aµM 6-well cell culture incubator. The next day, cells were treatedTHP-1 with either A), 0.16 PMA plate (Panel 5 B), PMA (Panel C) incubated and at the same time loaded Beads-Rabbit IgG-FITC Complex. at 5orx1.6 10µMcells/well and overnight in awith cellLatex culture incubator. The next day, The phagocytosis analyzed by (Panel fluorescence microscopy. PMA treatment a dose low cellsdegree wereoftreated with was either vehicle A), 0.16 µM PMA (Panel B), orat1.6 µM as PMA as 0.16 µM caused THP-1 cells to differentiate and become phagocytic, as evidenced by a significant (Panel C) and at the same time loaded with Latex Beads-Rabbit IgG-FITC Complex. The increase in cells engulfing the Latex Beads-Rabbit IgG-FITC Complex.

degree of phagocytosis was analyzed by fluorescence microscopy. PMA treatment at a dose as low as 0.16 µM caused THP-1 cells to differentiate and become phagocytic, as evidenced by a significant increase in cells engulfing the Latex Beads-Rabbit IgGFITC Complex.

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Figure 2. PMA increases phagocytosis in THP-1 cells as measured by flow cytometry. THP-1 cells were seeded in a 6-well plate at 5 x 105 cells/well and incubated overnight in a cell culture incubator. The next day, cells were treated with either vehicle (black line), 0.16 µM PMA (blue line), or 1.6 µM PMA (red line) and at the same time loaded with Latex Beads-Rabbit IgG-FITC Complex. The degree of phagocytosis was analyzed by an Accuri C6 flow cytometer. PMA treatment at a dose as low as 0.16 µM caused a significant increase in the number of phagocytic cells, as evidenced by a significant shift of the cell population to higher fluorescence.


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References

RESOURCES

1. Underhill, D.M. and Ozinsky, A. Phagocytosis of Microbes: Complexity in action. Annu. Rev. Immunol. 20, 825-852 (2002). 2. Beletski, A., Cooper, M., Sriraman, P., et al. High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye. Biotechniques 39, 894-897 (2005).

Troubleshooting Problem

Possible Causes

Recommended Solutions

Related Products

Cells do not respond to treatment

A. Cells are from a late passage and may have lost the capacity to respond B. Cells are not healthy

A. Use cells at a low passage number B. Use only healthy cells

High background staining in all cells regardless of treatment

A. Inadequate washing B. Cells used in the experiment have tendency to attract the bead complex to the membrane

A. Perform washes with Assay Buffer B. Use Trypan Blue included in the kit to quench nonspecific staining

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RESOURCES

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RESOURCES

RESOURCES

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G

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E

A

For further details, please refer to our Warranty and Limitation of Remedy located on our website and in our catalog.

D

Said refund or replacement is conditioned on Buyer giving written notice to Cayman within thirty (30) days after arrival of the material at its destination. Failure of Buyer to give said notice within thirty (30) days shall constitute a waiver by Buyer of all claims hereunder with respect to said material.

C

Buyer’s exclusive remedy and Cayman’s sole liability hereunder shall be limited to a refund of the purchase price, or at Cayman’s option, the replacement, at no cost to Buyer, of all material that does not meet our specifications.

B

Cayman Chemical Company makes no warranty or guarantee of any kind, whether written or oral, expressed or implied, including without limitation, any warranty of fitness for a particular purpose, suitability and merchantability, which extends beyond the description of the chemicals hereof. Cayman warrants only to the original customer that the material will meet our specifications at the time of delivery. Cayman will carry out its delivery obligations with due care and skill. Thus, in no event will Cayman have any obligation or liability, whether in tort (including negligence) or in contract, for any direct, indirect, incidental or consequential damages, even if Cayman is informed about their possible existence. This limitation of liability does not apply in the case of intentional acts or negligence of Cayman, its directors or its employees.

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Warranty and Limitation of Remedy

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NOTES

This document is copyrighted. All rights are reserved. This document may not, in whole or part, be copied, photocopied, reproduced, translated, or reduced to any electronic medium or machine-readable form without prior consent, in writing, from Cayman Chemical Company. ©05/02/2014, Cayman Chemical Company, Ann Arbor, MI, All rights reserved. Printed in U.S.A.

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RESOURCES