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Human Neutrophil Isolation Kit (Column-Based)

Human Neutrophil Isolation Kit (Column-Based)

Catalog Number: S0K1017 Reactivity: Human Conjugation: Brand: Starter
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$1,335 USD
$1,335 USD
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Product Details

Product Specification


Format

1 mL Human Neutrophil Biotin-Antibody Cocktail

1.5 mL Streptavidin Beads
Capacity

Suitable for a total cell count of 1×10⁹, and can perform up to 100 sorting runs.
Principle of Separation

This kit employs a column-based negative selection method to isolate human neutrophils from peripheral blood. In this negative selection process, non-human neutrophils are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. As a result, the unwanted cells become bound to the magnetic beads via the antibody‑antigen complex, while the target human neutrophils remain unlabeled. The cell suspension is subsequently loaded onto the separation column; under the influence of a magnetic field, the magnetically labeled non-target cells are retained within the column, whereas the unlabeled human neutrophils elute by gravity into the collection tube. The resulting supernatant represents the enriched fraction of human neutrophils after removal of the non-target cells.
Applications in cell sorting Label-free, native neutrophils can be isolated from peripheral blood of healthy donors and used for a variety of downstream functional assays.
Reactivity Human
Magnetic Bead Size 20 nm
Separation Method Column-Based
Selection Strategy Negative Selection
Stability & Storage

Store protected from light at 2–8 °C; do not freeze.

Background

Human neutrophils are the most abundant innate immune myeloid cells in peripheral blood, with a core phenotype of CD16+CD66b+. They constitute the body's first line of defense against bacterial and fungal infections and are extensively involved in key physiological and pathological processes, including inflammatory responses, immune regulation, tissue damage repair, autoimmune diseases, and the modulation of the tumor microenvironment. The isolation of high-quality, highly active, and highly pure primary neutrophils is a fundamental prerequisite for conducting cell function validation, studying inflammatory mechanisms, screening drugs, and advancing basic research in immunology.

This kit is developed based on immunomagnetic bead–based negative selection technology. It employs biotinylated antibodies to specifically label and deplete non-target cells from peripheral blood, including T cells, B cells, NK cells, monocytes, basophils, and red blood cells, without labeling or otherwise disturbing the target neutrophils throughout the process. The isolated cells can be directly used in a variety of downstream research applications, such as flow cytometry, cell activation and functional assays, inflammatory cytokine secretion analysis, and in vitro drug intervention experiments, providing a stable and reliable cell separation solution for studies of immune and inflammatory mechanisms.

Protocol

WithL Separation Columnas an example

Steps

Operation Instructions

Dosage and Time

Cell Processing and Labeling

1

Collect fresh human peripheral blood, and use Red Blood Cell Lysis Buffer (without fixative, 1X) (Cat: S0D9002) to lyse red blood cells; after preparing a single-cell suspension, proceed with cell counting;

Note:It is recommended to count cells after staining them with AO/PI.

Prepare the sample.

2

Resuspend 1×10^7 cells in 100 μL of MagSep Separation Buffer (Cat: S0D3018).7cells;

Note:Adjust reagent volumes proportionally based on sample size; set aside a pre-sorting sample to measure the proportion of human neutrophils.

1×10^77cells/100 μL

3

Add 10 μL of Human Neutrophil Biotin Antibody Cocktail to the sample and gently mix.

Note:In this step, gently pipette up and down 2–3 times to mix thoroughly;

10 μL/100 μL

4

Incubate the antibodies in the cocktail with the cells at 2–8℃ for 10 minutes;

Incubate at 2–8℃ for 10 minutes

5

Add MagSep Separation Buffer equal to ten times the cell volume to wash the cells, centrifuge at 400g for 7 minutes, discard the supernatant, and resuspend to the original volume;

Centrifuge at 400g for 7 minutes

6

Mix wellStreptavidin Beads, add 15 μLStreptavidin Beadsto the sample;

15 μL/100 μL

7

Gently mix the magnetic beads and cells, incubate at 2–8℃ for 5 minutes;

Incubate at 2–8℃ for 5 minutes

Cell sorting

8

In the column separation magnet, installL Separation Column, add 3 mL of MagSep Separation Buffer to wash the separation column;

Wash the separation column

9

Add the cells to the separation column and collect the target cells using a collection tube;

Note:The cell volume should be at least 1 mL; if insufficient, make up the volume with MagSep Separation Buffer; the cells that drip down under gravity are the target cells;

Collect the target cells

10

Add 3 mL of MagSep Separation Buffer three times, collecting the target cells each time;

Note:This step elutes the unlabeled target cells from the separation column, increasing the yield.

Collect the target cells

Notes: Pay attention to the maximum capacity of the separation column,The S Separation Column can handle a maximum of1×107cells;The L Separation Column has a maximum capacity of1×108;


Picture

Validation Data

Neutrophils were enriched from fresh human blood using a Human Neutrophil column-based negative selection kit. Cells were stained with CD66B-Alexa Fluor® 488 (SDT Cat: S0B8109) and CD16-Alexa Fluor® 647 (SDT Cat: S0B5525) fluorescent antibodies, and analysis was performed by gating on live cells (Live+), allowing for the determination of the proportion of neutrophils in the sorted fraction. Flow cytometry results indicated that the purity of neutrophils after sorting was 97.5%.