Mouse Neutrophil Cell Isolation Kit (Negative Selection), IC-1190
Product Introduction
This product is a negative selection-based immunomagnetic cell isolation kit specifically designed for the rapid and efficient enrichment of untouched, highly purified mouse neutrophils from bone marrow, spleen, or peripheral blood. Unlike positive selection methods that bind antibodies directly to neutrophils, this negative selection approach employs a cocktail of biotinylated monoclonal antibodies directed against cell surface markers expressed on non-neutrophil hematopoietic cells (e.g., T cells, B cells, NK cells, monocytes, erythrocytes, and other non-target lineages). Subsequent incubation with streptavidin-conjugated magnetic beads enables the depletion of unwanted cell populations using a magnetic separator, leaving the desired neutrophil population untouched and unlabeled in the supernatant. This negative selection strategy preserves the native functional status of isolated neutrophils, avoiding potential activation, receptor crosslinking, or signaling artifacts associated with antibody binding, making the isolated cells ideal for downstream functional assays including chemotaxis, phagocytosis, reactive oxygen species production, and in vivo adoptive transfer studies.
Product Features
1. Negative Selection Strategy: Isolates untouched neutrophils without antibody binding to the target cells, preserving native functional status and avoiding activation artifacts.
2. High Purity and Yield: Consistently achieves neutrophil purity of >90% from mouse bone marrow with excellent recovery rates and minimal contamination by other lineages.
3. Rapid and Simple Protocol: Complete isolation workflow achievable in 30-45 minutes using standard laboratory magnetic separation equipment.
4. Versatile Sample Compatibility: Validated for isolation of neutrophils from mouse bone marrow, spleen, and peripheral blood samples.
5. Ready-to-Use Kit Format: Includes all necessary antibody cocktail, magnetic beads, and buffers for a complete cell isolation workflow.
Specifications
Size: 50 isolations
Sample Sources: Mouse bone marrow, spleen, peripheral blood
Isolation Method: Negative selection (immunomagnetic depletion)
Target Cells: Untouched mouse neutrophils
Purity: Typically >90%
Cell Viability: >95%
Equipment Required: Magnetic separator (e.g., EasySep™ magnet, MACS® separator, or equivalent)
Storage and Stability
Storage Conditions: Store all kit components at 4°C. Do not freeze. Protect from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. After opening, use within 6 months when handled aseptically.
Protocol (For Reference Only)
Important: Use aseptic technique throughout the isolation procedure. Work quickly and keep cells and reagents at 4°C to maintain viability and minimize non-specific binding. All centrifugation steps should be performed at 4°C with pre-cooled buffers.
1. Sample Preparation: Prepare a single-cell suspension from mouse bone marrow (by flushing femurs and tibias), spleen (by mechanical dissociation and filtration), or peripheral blood (by density gradient centrifugation or red blood cell lysis). Filter the cell suspension through a 70 µm cell strainer to remove debris and cell aggregates. Count cells and assess viability by trypan blue exclusion.
2. Cell Washing and Resuspension: Centrifuge the cell suspension at 300 x g for 5 minutes at 4°C, discard the supernatant, and resuspend the cell pellet in cold Isolation Buffer at a concentration of 1×10⁸ cells/mL. Transfer the cell suspension to a 5 mL round-bottom polystyrene tube (compatible with the magnetic separator being used).
3. Antibody Cocktail Incubation: Add the provided Biotinylated Antibody Cocktail to the cell suspension at the recommended volume (typically 50 µL per 1×10⁸ total cells). Mix gently by pipetting and incubate at 4°C for 15 minutes. Do not vortex.
4. Magnetic Bead Incubation: After antibody incubation, add the provided Streptavidin Magnetic Beads to the cell suspension at the recommended volume (typically 50 µL per 1×10⁸ total cells). Mix gently by pipetting and incubate at 4°C for 15 minutes.
5. Magnetic Separation: Add Isolation Buffer to bring the total volume to 2.5 mL. Gently mix by pipetting, then place the tube in the magnetic separator without the cap. Incubate at room temperature for 5-10 minutes to allow complete magnetic separation. While the tube remains in the magnet, carefully pour off or aspirate the supernatant containing the enriched untouched neutrophils into a clean 15 mL conical tube. Do not disturb the magnetically bound unwanted cells adhering to the tube wall.
6. Post-Isolation Processing: Centrifuge the collected neutrophil suspension at 300 x g for 5 minutes at 4°C. Resuspend the purified neutrophil pellet in appropriate medium (e.g., RPMI-1640, HBSS, or PBS) for downstream functional assays or in vivo administration. Assess purity by flow cytometry using neutrophil markers (Ly6G⁺CD11b⁺) and viability by 7-AAD or PI staining.
Precautions
1. All steps should be performed at 4°C with pre-cooled buffers to maintain cell viability and prevent neutrophil activation.
2. Use the magnetic separator recommended for this kit; different magnet designs may require optimization of incubation times and separation volumes.
3. Do not vortex cells after addition of antibody cocktail or magnetic beads; gentle pipetting is sufficient for mixing.
4. Neutrophils are short-lived primary cells; process samples promptly and use isolated cells within 2-4 hours for optimal functional activity.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the advantage of negative selection over positive selection for neutrophil isolation?
A1: Negative selection leaves neutrophils untouched and unlabeled, avoiding potential functional alterations caused by antibody binding to neutrophil surface receptors. This is critical for downstream assays where receptor signaling, activation status, or in vivo trafficking behavior must remain unperturbed.
Q2: What purity and yield can I expect from this kit?
A2: Typical purity of isolated neutrophils is >90% as assessed by Ly6G and CD11b flow cytometric staining, with yields of approximately 5-10×10⁶ neutrophils from one mouse femur, depending on mouse strain, age, and health status.
Q3: Can I use this kit for human neutrophil isolation?
A3: No. This kit is specifically designed for mouse neutrophils. The antibody cocktail targets mouse-specific cell surface markers. For human neutrophil isolation, a species-specific kit should be used.
Q4: What functional assays can be performed with the isolated neutrophils?
A4: Isolated untouched neutrophils are suitable for a wide range of functional assays including chemotaxis (Boyden chamber or Transwell), phagocytosis, reactive oxygen species production, degranulation, NETosis, and in vivo adoptive transfer or trafficking studies.
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 isolation conditions may be required for specific mouse strains or tissue sources.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product and animal tissues.
Ordering Information
Catalog Number: IC-1190
Product Name: Mouse Neutrophil Cell Isolation Kit (Negative Selection)
Size: 50 isolations
Price: CNY ¥5150.00 / USD $515.00 / EUR �618.00 / JPY ¥92700.00
2023 Version
