Apoptosis and Necrosis Detection Kit, Three-Color Fluorescence, IC-6503

Product Introduction

This product is a tool kit for monitoring cell viability through multiple parameters. It is designed for the simultaneous detection of apoptotic cells, necrotic cells, and healthy cells.

Apoptosis is an active, programmed process of autonomous cellular disassembly that does not provoke an inflammatory response. During apoptosis, phosphatidylserine (PS) is externalized to the outer leaflet of the cell membrane. As a universal marker of early to mid-stage apoptosis, PS exposure can be detected even before morphological changes occur. The PS probe used in this kit emits green fluorescence upon binding to PS on the membrane surface.

Necrosis is a passive, accidental form of cell death caused by environmental perturbation, accompanied by uncontrolled release of inflammatory cellular contents. Staining of the nucleus with the membrane-impermeable dye 7-AAD (Ex/Em = 546/647 nm) provides a direct indication of loss of cell membrane integrity, a characteristic of late apoptosis and necrosis.

This kit also provides Violet 450 (Ex/Em = 405/450 nm), a live-cell cytoplasmic labeling dye for staining the cytoplasm of viable cells.

The kit is optimized for simultaneous detection of the following by flow cytometry or fluorescence microscopy:

Apoptotic cells (green)

Necrotic cells (green and/or red)

Healthy cells (blue)

Product Features

1. Three-Color Detection: Simultaneous detection of apoptotic, necrotic, and healthy cells in a single sample.

2. Multi-Platform Compatibility: Suitable for both flow cytometry and fluorescence microscopy.

3. Optimized Formulation: All reagents optimized for reproducible staining and minimal spectral overlap.

4. Complete Kit Format: Includes PS probe, 7-AAD, live-cell cytoplasmic dye, and assay buffer.

Specifications

Size: 100 tests

Components: Green (100X), Assay Buffer, 7-AAD (200X), Violet 450 (200X)

Detection Method: Flow cytometry or fluorescence microscopy

Application: Apoptosis and necrosis detection

Instrument Compatibility

Flow Cytometry:

Excitation: 405 nm, 488 nm

Emission: 450¡À20 nm, 530nm¡À15nm, 670¡À7 nm

Channels: Pacific Blue, FITC, PE-Cy5

Fluorescence Microscopy:

Excitation/Emission: DAPI, FITC, Texas Red filter sets

Recommended Plate: Black-walled, clear-bottom plate

Storage and Stability

Storage Conditions: Store all components at -20¡ãC, protected from light. Aliquot reconstituted solutions into single-use volumes to avoid repeated freeze-thaw cycles.

Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed.

Protocol (For Reference Only)

Important: Protect fluorescent reagents from light throughout the procedure. Prepare working solutions fresh before use.

Working Solution Preparation:

Violet 450 Working Solution (200X): Add 100 µL DMSO to the Violet 450 vial (Component D) to prepare a 200X working solution. Store protected from light.

Experimental Procedure:

1. Treat cells with test compounds for the desired time (e.g., Jurkat cells treated with staurosporine for 4-6 hours).

2. Collect cells by centrifugation and adjust density to 1-5¡Á10⁵ cells per tube.

3. Resuspend cells in 200 µL Assay Buffer (Component B).

4. Add 2 µL of 100X Green (Component A).

Optional Step 1: To detect necrotic cells, add 1 µL of 200X 7-AAD (Component C).

Optional Step 2: To detect healthy cells, add 1 µL of 200X Violet 450 working solution.

5. Incubate at room temperature for 30-60 minutes protected from light.

6. Add 300 µL Assay Buffer (Component B) to adjust sample volume.

7. Analyze by flow cytometry or fluorescence microscopy.

Apoptotic cells: 530¡À15nm emission filter (FITC channel)

Healthy cells: 450¡À20 nm emission filter (Pacific Blue channel)

Necrotic cells: 670¡À7 nm emission filter (PE-Cy5 channel)

Flow Cytometry Analysis:

Quantify Green binding using a flow cytometer equipped with 530¡À15nm (FITC channel - apoptotic cells), 450¡À20 nm (Pacific Blue channel - healthy cells), and 670¡À7 nm (PE-Cy5 channel - necrotic cells) emission filters.

Note: Due to potential membrane damage during cell dissociation or collection, flow cytometric analysis of in adherent cells has not been established as a routine assay. However, flow cytometric detection of Annexin V in adherent cells has been previously reported.

Fluorescence Microscopy Analysis:

After incubation, aspirate the cell suspension, wash 1-2 times with Assay Buffer, and resuspend cells in Assay Buffer. Apply cells to a coverslip or black-walled/clear-bottom 96-well plate.

Note: For adherent cells, culture cells directly on coverslips or black-walled/clear-bottom 96-well plates. After co-incubation with Green, wash 1-2 times with Assay Buffer, then add Assay Buffer to cover the coverslip or plate. Invert the coverslip onto a slide for observation. Alternatively, fix cells with 2% formaldehyde after Green incubation for microscopy observation.

Observe Green-labeled apoptotic cells using a FITC filter set. When 7-AAD is added, use a Texas Red filter set to assess cell viability. When  Violet 450 is added, use a DAPI or violet filter set. Green fluorescence on the cell membrane surface indicates Green binding to phosphatidylserine (PS).

Precautions

1. Protect fluorescent reagents from light throughout the procedure.

2. Prepare working solutions fresh before use.

3. Aliquot reconstituted solutions into single-use volumes to avoid repeated freeze-thaw cycles.

4. For research use only. Not for use in diagnostic or therapeutic procedures.

FAQ (Simplified)

Q1: What is the detection principle of this kit?

A1: The kit uses a green fluorescent PS probe (Green) to detect apoptotic cells, 7-AAD to detect necrotic cells with compromised membrane integrity, and  Violet 450 to label the cytoplasm of healthy cells.

Q2: Can this kit be used with adherent cells?

A2: Yes. For microscopy analysis, culture adherent cells directly on coverslips or black-walled/clear-bottom plates. For flow cytometry, dissociation may cause membrane damage; validation for specific cell types is recommended.

Q3: How should I store the kit components?

A3: Store at -20¡ãC protected from light. Aliquot reconstituted solutions into single-use volumes to avoid repeated freeze-thaw cycles.

Disclaimer

1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

2. Due to the variable nature of biological research, optimization of staining conditions is recommended for specific cell types.

3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages.

4. Wear appropriate protective clothing and gloves when handling this product.

Ordering Information

Catalog Number: IC-6503

Product Name: Apoptosis and Necrosis Detection Kit, Three-Color Fluorescence

Size: 100 tests

Suggested Retail Price: CNY ¥3550.00 / USD $355.00 / EUR €426.00 / JPY ¥63900.00

Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.