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Human Pan Monocyte Cell Isolation Kit (Column-Based)

Human Pan Monocyte Cell Isolation Kit (Column-Based)

Catalog Number: S0K1012 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

1 mL 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-Pan monocytes (CD14+CD16+) are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. As a result, the unwanted cells are marked by the antibody–bead complex, while the target Pan monocytes (CD14+CD16+) remain unlabeled. The cell suspension is subsequently transferred to a separation column; under the influence of a magnetic field, the magnetically labeled non-target cells adhere to the column, whereas the unlabeled Pan monocytes (CD14+CD16+) elute into a collection tube under gravity. The resulting supernatant represents the enriched fraction of Pan monocytes (CD14+CD16+) after removal of the non-target cells.
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

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 inflammatory responses, 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 remove contaminating cells such as lymphocytes, granulocytes, and erythrocytes, thereby efficiently enriching human CD14+CD16+ positive pan‑monocytes. The sorting process is gentle, avoiding cell activation and preserving surface markers, thus fully maintaining the cells' native 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 the separation buffer; you will need to provide it yourself. The corresponding Stater catalog number isS0D3018

Taking the L Separation Column as an example

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Isolate PBMCs from peripheral blood using density gradient centrifugation with lymphocyte separation medium, or directly thaw cryopreserved PBMC cells, and then perform cell counting.

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

Prepare the sample

2

Resuspend 1×10 in 100 μL of MagSep Separation Buffer7cells;

Note:Adjust reagents proportionally according to the sample volume; set aside a pre-sorting sample to test the proportion of Human Pan Monocyte cells;

1×107cells/100 μL

3

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

5μL/100 μL

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

Note:For this step, gentle pipetting to mix 2–3 times is recommended.

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

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

Centrifuge at 400g for 7 minutes

7

Mix wellStreptavidin Beads, add 10 μLStreptavidin Beadsto the sample;

10 μL/100 μL

8

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

Incubate at room temperature for 5 minutes

Cell sorting

9

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

Wash the separation column

10

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

Note:The cell volume should be at least 500 μL; 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

11

Add 3 mL of MagSep Separation Buffer and collect the target cells;

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 hold a maximum of1×107cells;The L Separation Column has a maximum capacity of1×108;


Picture

Validation Data

Human Pan Monocytes were enriched from peripheral blood mononuclear cells (PBMCs), and the sorted cells were stained with CD14-Alexa Fluor® 488 (SDT Cat: S0B8022) and CD16-Alexa Fluor® 647 (SDT Cat: S0B85525). Analysis was performed by gating on live cells (Live+). Flow cytometric results showed that the purity of the sorted Human Pan Monocytes (CD14+CD16+) was 90.95%.