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Human B Cell Isolation Kit (Column-Based)

Human B Cell Isolation Kit (Column-Based)

Catalog Number: S0K1013 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Human B Cell Biotin-Antibody Cocktail

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

This kit employs a column-based negative selection method to isolate B cells from human peripheral blood mononuclear cells (PBMCs). In this negative-selection process, non-B cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. As a result, the non-target cells become bound to the magnetic beads via the antibody‑antigen complex, while the target B cells remain unlabeled. The cell suspension is subsequently loaded onto a separation column; under the influence of a magnetic field, the magnetically labeled non-target cells are retained within the column, whereas the unlabeled B cells elute by gravity into a collection tube. The resulting supernatant constitutes the B-cell‑enriched fraction 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

Human B lymphocytes are the core effectors of adaptive humoral immune responses, performing critical functions such as antigen recognition, processing and presentation, antibody secretion, and maintenance of immunological memory. They play an irreplaceable role in defending against extracellular pathogen infections and regulating the magnitude of immune responses. The CD19 molecule is the most representative lineage-specific marker on the B-cell surface; it belongs to the immunoglobulin superfamily and is stably expressed from the early pre-B cell stage through to mature plasma cells. CD19 collaborates with the B-cell receptor to regulate B-cell activation, proliferation, and differentiation, serving as a crucial regulatory molecule for B-cell development and functional maintenance. Consequently, it is widely used as a specific surface antigen of human B cells. In immunological research, obtaining B cells that are highly pure, highly active, and have intact, native surface molecules is an essential prerequisite for ensuring the reliability of experimental results. This negative-selection kit is based on the principle of negative sorting, employing a biotin‑labeled antibody panel to specifically label non‑B cells. The identified targets include various interfering cell populations, such as T cells, NK cells, monocytes, macrophages, platelets, and erythroid cells. Streptavidin‑coated nanomagnetic beads are then used to efficiently remove non‑target cells. Throughout the entire sorting process, no antibodies bind to any antigens on the B‑cell surface, thereby maximizing the preservation of the cells' original morphology, the integrity of their surface markers, and their biological functional activity, providing high‑quality cellular material for downstream applications such as gene expression analysis, in vitro culture and expansion, and functional characterization.

Protocol

Note: This kit does not include the separation buffer; it must be provided separately. The corresponding Stater catalog number isS0D3018

TakingL Separation Columnas 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 solution, 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 1×10^7 cells in 100 μL of MagSep Separation Buffer.7cells;

Note:Adjust reagent volumes proportionally based on sample volume; set aside a pre-sorting sample to determine the proportion of human B cells.

1×107cells/100 μL

3

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

Note:In this step, gently pipette the mixture 2–3 times to mix thoroughly;

10 μL/100 μL

4

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

Incubate at room temperature for 10 min

5

Mix wellStreptavidin Beads, add 10 μLStreptavidin Beadsinto the sample;

10 μL/100 μL

6

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

Incubate at room temperature for 5 min

7

Place it in the column selection magnet,L Separation Columnadd 3 mL of MagSep Separation Buffer to rinse the separation column;

Rinse the separation column

Cell sorting

8

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, add MagSep Separation Buffer to make up the volume. The cells that drip down under gravity are the target cells.

Collect the target cells

9

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: 1.Pay attention to the maximum capacity of the separation column.The S Separation Column can hold a maximum of1×107;the L Separation Column's maximum labeled cell count is1×108;

2.If the sample viability is low, it is recommended to use a dead cell removal kit to eliminate dead cells (Dead Cell Removal Set, (RUO)#S0K0009);


Picture

Validation Data

Human B cells were enriched from peripheral blood mononuclear cells (PBMCs), and the sorted cells were stained with CD19-APC (clone: HIB19) and CD3-FITC (clone: UCHT1) fluorescent antibodies. Analysis was performed by gating on live cells (Live+), and the proportion of B cells in the sorted fraction was determined. Flow cytometric analysis revealed that the purity of B cells after sorting was 97.8%.