Human CD66b+Cell Sorting Kit (Positive Selection)

Cat. No.: H1522391

GRADE & PURITY BioReagent

Overview

The Human CD66b⁺ Cell Isolation Kit (Positive Isolation) is suitable for isolating CD66b⁺ cells from a single-cell suspension of human peripheral blood. The principle is to label CD66b⁺ cells using a CD66b capture antibody, then capture the target cells using releasable magnetic beads. The beads are subsequently dissociated from the cell surface using a Magnetic Beads Release Buffer, yielding human CD66b⁺ cells free of magnetic bead labeling. CD66b is a 95-100 kDa glycosylphosphatidylinositol (GPI)-linked protein that is expressed on neutrophils and eosinophils, but not on basophils or lymphocytes. The isolated CD66b⁺ cells can be used in downstream molecular biology and cell biology experiments.

Scope of Application

Isolation of CD66b⁺ cells from human peripheral blood.

H1522391	Component	Appearance	10T	100T	Storage
H1522391A	CD66b Capture Antibody	Liquid	20 μL	200 μL	2-8℃.
H1522391B	Releasable Magnetic Beads	Liquid	200 μL	1 mL ×2	2-8℃.
H1522391C	Magnetic Beads Release Buffer	Liquid	4 mL	40 mL	2-8℃.

Cell Sample Preparation Procedure

1. Red Blood Cell Lysis

Place fresh anticoagulated whole blood into a centrifuge tube and lyse the red blood cells.

Note: EDTA-anticoagulated whole blood is recommended. Adjust the volume and duration of the red blood cell lysis step according to the lysis buffer used. If lysis is insufficient, repeat the lysis step. The presence of a small number of residual red blood cells will not affect the purity of the isolated cells.

2. Cell Washing

After lysis, resuspend the cells in PBS, wash, and centrifuge at 500 × g for 5 minutes.

3. Adjust Cell Density

After centrifugation, discard the supernatant and resuspend the cells in Isolation Buffer. Adjust the cell density to 1 × 10⁸ cells/mL.

Note: Isolation Buffer is PBS containing 2 mM EDTA and 2% fetal bovine serum (FBS). It must be pre-filtered through a 0.22 μm filter for sterilization.

Cell Isolation Procedure

1. Add CD66b Capture Antibody

Add 500 μL of cell suspension (5 × 10⁷ cells) to the bottom of a sterile flow cytometry tube. Then add 10 μL of CD66b Capture Antibody. Mix well and incubate at 4°C for 15 minutes.

Note: Add the cell suspension directly to the bottom of the flow tube, avoiding adding it along the tube wall. Depending on the magnetic stand used, a centrifuge tube can also be used for cell isolation. For isolating other numbers of cells, scale the volume of CD66b Capture Antibody proportionally. If isolating fewer than 1 × 10⁷ cells, bring the cell suspension volume to 100 μL and add 2 μL of CD66b Capture Antibody.

2. Add Releasable Magnetic Beads

After incubation, add 100 μL of washed Releasable Magnetic Beads to the flow tube. Mix well and incubate at 4°C for 15 minutes (Beads need to be washed before use: vortex to resuspend the beads, transfer the required volume to a 1.5 mL centrifuge tube, add 1 mL of Isolation Buffer, centrifuge at 10,000 × g for 1 minute, or use a magnetic stand for 3 minutes, and discard the supernatant. Add 1 mL of Isolation Buffer and repeat the wash. After the second wash, resuspend the beads in the original volume of Isolation Buffer. For example, if 20 μL of beads are washed, resuspend them in 20 μL of Isolation Buffer).

Note: For isolating other numbers of cells, scale the volume of Releasable Magnetic Beads proportionally. If isolating fewer than 1 × 10⁷ cells, use 20 μL of Releasable Magnetic Beads.

3. First Magnetic Separation

After incubation, add Isolation Buffer to the flow tube to bring the volume to 2.5 mL. Mix thoroughly by pipetting (avoid inverting the tube). Place the flow tube on a magnetic stand and let stand for 5 minutes.

4. Discard Supernatant and Resuspend Beads

Aspirate and discard the supernatant. Remove the flow tube from the magnetic stand, quickly add 2 mL of Isolation Buffer, and pipet repeatedly to disperse the beads. Place the tube back on the magnetic stand and let stand for 5 minutes.

5. Repeat Washing

Repeat Step 4 twice (thorough washing ensures high purity of target cells after subsequent elution).

6. First Elution

After the final magnetic aspiration, aspirate and discard the supernatant. Remove the flow tube from the magnetic stand, quickly add 1 mL of Magnetic Beads Release Buffer, and thoroughly resuspend the beads. Avoid allowing the beads to dry out. Transfer the bead suspension to a 1.5 mL centrifuge tube and incubate with rotation at room temperature for 10 minutes.

Note: For isolating other numbers of cells, scale the volume of Magnetic Beads Release Buffer proportionally. If isolating fewer than 1 × 10⁷ cells, use 200 μL of Magnetic Beads Release Buffer for cell elution.

7. Collect First Eluted Cells

After incubation, pipet repeatedly at least 10 times. Transfer the bead suspension to a new flow tube, place it on a magnetic stand, and let stand for 5 minutes.

Note: When isolating a smaller number of cells, add Isolation Buffer to bring the volume to 1.5 mL before magnetic separation. For example, for 2 × 10⁷ cells (using 400 μL of Magnetic Beads Release Buffer), after transferring to a new flow tube, add Isolation Buffer to 1.5 mL, pipet to mix, then place on the magnetic stand.

8. Second Elution

Transfer the supernatant to a new flow tube and set aside (the supernatant contains the target cells; do not discard). Quickly resuspend the beads in 1 mL of Magnetic Beads Release Buffer, avoiding drying. Transfer the bead suspension to a 1.5 mL centrifuge tube and incubate with rotation at room temperature for 10 minutes.

9. Collect Second Eluted Cells

After incubation, pipet repeatedly at least 10 times. Transfer the bead suspension to a new flow tube, place on a magnetic stand, and let stand for 5 minutes.

10. Remove Residual Beads

Combine the supernatant with the supernatant from the first elution (from Step 7). Place the tube on a magnetic stand and let stand for 5 minutes to remove any residual beads.

11. Collect Cells by Centrifugation

Transfer the supernatant to a centrifuge tube and centrifuge at 500 × g for 5 minutes. Discard the supernatant to collect CD66b⁺ cells free of magnetic bead labeling.

Isolation Performance

CD66b⁺ cells were isolated from human peripheral blood cells. The cells were stained with AF 647-conjugated anti-CD66b antibody (clone 6/40c) and analyzed by flow cytometry. The purity of CD66b⁺ cells before and after isolation was 52.62% and 99.67%, respectively.

Precautions

1. Avoid freezing all components of the kit during use and storage.

2. Low-retention pipette tips and centrifuge tubes are recommended to avoid loss of magnetic beads and antibodies due to adsorption.

3. This product must be used in conjunction with a magnetic stand.

4. This product is for research use only.

Specifications

Specifications & Purity

BioReagent

Grade

BioReagent

Application

Cell analysis

Storage and Shipping

Storage

Store at 2-8°C,Do not freeze

Shipped In

Wet ice,Do not freeze

Stability And Storage

Store at 2-8℃ long term (12 months). Do not freeze.

Contents & Storage

H1522391	Component	Appearance	10T	100T	Storage
H1522391A	CD66b Capture Antibody	Liquid	20 μL	200 μL	2-8℃.
H1522391B	Releasable Magnetic Beads	Liquid	200 μL	1 mL ×2	2-8℃.
H1522391C	Magnetic Beads Release Buffer	Liquid	4 mL	40 mL	2-8℃.