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Human Neutrophil Isolation Kit (Column-Free)

Human Neutrophil Isolation Kit (Column-Free)

Catalog Number: S0K3016 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

1.5mL Streptavidin Beads
Capacity

Applicable to1×10⁹ total cell count, and can perform up to100sorts.
Principle of Separation

In this negative selection process, biotin-conjugated antibodies are used to label non-target cells.HumanNeutrophilcells, and then the cells are incubated with streptavidin-coated nanomagnetic beads. Non-target cells are labeled by the antibody‑bead complex, while the targetHuman Neutrophilcells remain unlabeled. Subsequently, the cell suspension is transferred to a polystyrene tube and placed within the magnetic field of the sorter. Under the influence of the magnetic field, the magnetically labeled non-target cells adhere to the tube wall, whereas the unlabeledHuman Neutrophilcells remain in the supernatant. This supernatant is the fraction ofHumanNeutrophilcell-enriched component after removing 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 150 nm
Separation Method Column-Free
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 ofCD16+CD66b+. They constitute the body's first line of defense against bacterial and fungal infections, while also extensively participating in inflammatory responses, immune regulation, tissue damage repair, autoimmune diseases, and the regulation of the tumor microenvironment—key physiological and pathological processes. The isolation of high-quality, highly active, and high-purity primary neutrophils is a fundamental prerequisite for conducting cell function validation, studying inflammatory mechanisms, drug screening, and basic research related to immunity.

This kit is developed based on immunomagnetic bead negative selection technology, using biotinylated antibodies to specifically label and remove from peripheral bloodTcells,Bcells,NKcells, monocytes, basophils, and red blood cells—non-target cells—throughout the entire process without labeling or touching the target neutrophils..The isolated cells can be directly used in a variety of downstream research applications, including flow cytometry, cell activation and functional assays, detection of inflammatory cytokine secretion, and in vitro drug intervention experiments, providing a stable and reliable cell isolation solution for studies on immune-inflammatory mechanisms.

Protocol

Steps

Operating Instructions

Dosage and Timing

1

Collect fresh human peripheral blood, requiring the use of red blood cell lysis solutionRed Blood Cell Lysis Buffer (without fixative, 1X)(Cat: S0D9002) for erythrocyte lysis treatment;After preparing a single-cell suspension, perform cell counting;

Note:It is recommended to count cells after staining withAO/PI;;

Prepare the sample

2

Use100 μLMagSep Separation Buffer(Cat: S0D3018) to resuspend1×107cells;

Note:Adjust reagents proportionally according to sample volume; Retain a pre-sorting sample for testingHuman Neutrophilratio;

1×107cells/100 μL

3

Add10 μLHuman NeutrophilBiotin Antibody Cocktailto the sample, and gently mix;

Note:In this step, gentle pipetting can be used to mix evenly2-3times;

10 μL/100 μL

4

Incubate the antibodies and cells in the cocktail, incubate at 2-8℃ for10 min;incubate at 2-8

℃ for10 min10 min

5

shakevortexfor 5–30 sto mix wellStreptavidin beads;

mix wellfor 5–30 s

6

add15 μL of Streptavidin beadsto the sample;

15 μL/100 μL

7

gently mix the magnetic beads and cells,at 2-8℃incubatefor5min

;Note:In this step, mixing can be done by gently pipetting up and down2–3

times;

In this step, the magnetic beads may settle at the bottom, so you can gently tap the tube twice to mix them;2-8℃incubatefor5

min

;8add

Add MagSep Separation Bufferto a total volume of2.5 mL(a5 mL flow cytometry tube)

Add the total volume to7.5 mL(15 mLcentrifuge tube)

9

Place the sample on the magnetic stand so that the magnetic beads adhere to the tube wall;

Adsorb using a single-well magnetic stand (3min)

Adsorb using a multi-well magnetic stand (5min)

10

Tilt the magnetic stand and pour the sample into a new collection tube to collect the sample;

Note:The sorted cells can be subjected to400gcentrifugationfor 7 minutes, for subsequent culture and analysis.

Cell sorting successful

Note:0.1-1 × 108cells(0.1-1 mL) are recommended to use5 mLsingle-well magnetic separator(Starter EasyIso Separator)with a total adsorption volume of2.5 mL;

2-5 × 108cells(2-5 mL) are recommended to use15 mLmulti-well magnetic separator(Starter EasyEights EasyIso Separator)with a total adsorption volume of7.5 mL;


Picture

Validation Data

Neutrophils were enriched from fresh human blood using the Human Neutrophil column-free 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 this sorting step was 97.1%.