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Human Pan Monocyte Cell Isolation Kit (Column-Free)

Human Pan Monocyte Cell Isolation Kit (Column-Free)

Catalog Number: S0K3010 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Human Pan Monocyte Cell Biotin-Antibody Cocktail

0.5 mL Human FcR

1mL Streptavidin Beads
Capacity

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

In this negative selection process, non-human pan monocytes (CD14+CD16+) are labeled with biotin-conjugated antibodies. The cells are then incubated with streptavidin-nanomagnetic beads; the non-target cells become bound to the beads via the antibody‑bead complex and are thus labeled, while the target human pan monocytes (CD14+CD16+) 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 unlabeled human pan monocytes (CD14+CD16+) remain in the supernatant. This supernatant constitutes the enriched fraction of human pan monocytes (CD14+CD16+) after removal of the non-target cells.
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

Monocytes are an important myeloid cell subset of the innate immune system, capable of differentiating into macrophages and dendritic cells. They participate in physiological and pathological processes such as the body's inflammatory response, antigen presentation, immune regulation, and tissue repair, making them a commonly used research subject for investigating immunological mechanisms, conducting cellular function experiments, and establishing inflammation models. Based on differences in the expression of CD14 and CD16 molecules on their surface, human monocytes can be classified into classical, intermediate, and non-classical subpopulations. CD14+CD16− monocytes constitute a key functional subtype, extensively involved in inflammatory responses, antigen presentation, immune regulation, and pathogen clearance, and thus hold significant research value in studies related to infection, autoimmune diseases, and tumor immunity.

This reagent employs immunomagnetic bead-based cell sorting to specifically deplete contaminating cells such as lymphocytes, granulocytes, and erythrocytes, thereby efficiently enriching human CD14+CD16+ positive classical monocytes. The sorting process is gentle, avoiding cell activation and preserving surface markers, thus fully maintaining the cells' intrinsic biological characteristics and physiological activity. The isolated cells exhibit high purity and stable viability, making them suitable for applications including cell subtype identification, polarization induction, cytokine secretion assays, in vitro functional validation, and investigations into disease mechanisms. They are well‑suited for basic immunological research and preclinical experimental settings.

Protocol

Note: This kit does not include sorting buffer; please provide it separately. The corresponding Stater catalog number isS0D3018

Steps

Operating Instructions

Dosage and timing

1

Peripheral blood mononuclear cells (PBMCs) are isolated from peripheral blood by density-gradient centrifugation using lymphocyte separation medium, or cryopreserved PBMCs are directly thawed and resuspended, followed by cell counting.

Note: It is recommended to count cells after staining with AO/PI; PBMCs can be centrifuged at 400g for 7 minutes.

Prepare the sample

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer.sevencell;

Note:Reagents are adjusted proportionally based on the sample volume; retain a pre-sorting sample for assessing the proportion of human pan-monocytes.

1×10sevencells/100 μL

3

Add 5 μL of Human FcR to the sample and gently mix.

5 μL/100 μL

4

Add 10 μL of the Human Pan Monocyte cell Biotin Antibody Cocktail to the sample and gently mix.

Note:For this step, gentle pipetting can be used to mix by blowing the solution up and down 2–3 times;

10 μL/100 μL

5

Incubate the antibodies in the cocktail with the cells at room temperature for 10 minutes;

Incubate at room temperature for 10 minutes

6

Shake the Streptavidin beads for 5–30 seconds to mix them evenly;

Shake for 5–30 seconds

7

Add 10 μL of Streptavidin beads to the sample;

10 μL/100 μL

8

Gently mix the magnetic beads and cells, and incubate at room temperature for 5 minutes;

Note:In this step, you can use a pipette to gently blow the mixture up and down 2–3 times;

During this step, the magnetic beads may settle at the bottom; you can mix again after about 2.5 minutes;

Incubate at room temperature for 5 minutes

9

Add MagSep Separation Buffer to the sample and immediately place it on the magnetic stand to prevent the magnetic beads from settling at the bottom in large quantities.

Adjust the total volume to 2.5 mL (in a 5 mL flow cytometry tube).

Adjust the total volume to 7.5 mL (in a 15 mL centrifuge tube).

10

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

Adsorb using a single-well magnetic rack (3 min).

Adsorb using a multi-well magnetic rack (5 min).

11

Tilt the magnetic rack and pour the sample into a new collection tube to collect the sample.

Note:Sorted cells can be centrifuged at 400g for 7 minutes, for subsequent culture and analysis.

Cell sorting successful

Note:For 0.1–1 × 10^8 cells (0.1–1 mL), it is recommended to use a 5 mL single-well magnetic stand (Starter EasyIso Separator) with a total adsorption volume of 2.5 mL.

2–5 × 10^8 cells (2–5 mL) are recommended to be adsorbed using a 15 mL row-type magnetic separator (Starter EasyEights EasyIso Separator), with a total volume of 7.5 mL.


Picture

Validation Data

Human Pan Monocyte cells were enriched from peripheral blood mononuclear cells (PBMCs). After sorting, the cells were stained with CD14-Alexa Fluor® 488 (SDT Cat: S0B8022) and CD16-Alexa Fluor® 647 (SDT Cat: S0B85525), and analysis was performed by gating on live cells (Live+). Flow cytometry results showed that the purity of the sorted Human Pan Monocyte (CD14+CD16+) cells was 92.87%.