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Mouse Pan B Cell Isolation Kit (Column-Based)

Mouse Pan B Cell Isolation Kit (Column-Based)

Catalog Number: S0K1010 Reactivity: Mouse Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse Pan 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 Pan B cells from mouse spleens. 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 unwanted 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 represents the B‑cell‑enriched fraction after removal of the non‑target cells.
Reactivity Mouse
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

Mouse B lymphocytes are the core cell population of the adaptive humoral immune response, performing crucial functions such as antigen presentation, antibody secretion, and the formation of immunological memory. They play an irreplaceable role in defending against extracellular pathogen infections and maintaining immune homeostasis. The CD19 molecule is the most representative lineage-specific marker on the surface of mouse B cells; it belongs to the immunoglobulin superfamily and is stably expressed from the early pre-B cell stage through the mature plasma cell stage. CD19 forms a signal-transduction complex with molecules such as CD21 and CD81, working synergistically with the B cell receptor (BCR) to lower the activation threshold of B cells and amplify downstream signaling cascades. As an important regulatory molecule in B cell development, activation, and differentiation, CD19 is widely used as a specific surface antigen for pan‑B cells in mice. In immunological research, obtaining highly purified, high‑activity pan‑B cells is a prerequisite for functional analysis and studies of signaling pathways. This negative selection kit is based on the principle of negative sorting, using a biotin‑labeled antibody cocktail (excluding anti‑CD19 antibodies) to specifically label non‑B cells (such as T cells, NK cells, macrophages, and granulocytes). Subsequently, streptavidin‑coated nanomagnetic beads efficiently remove these non‑target cells. Throughout the process, no antigens on the B cell surface are contacted, thereby maximizing the preservation of the cells' native morphology, the integrity of surface markers, and their physiological functional activity, providing high‑quality cellular material for downstream experiments.

Protocol

Note: This kit does not include separation buffer; please prepare it yourself. The corresponding Stater catalog number isS0D3018

TakingL Separation Columnas an example

Steps

Operating instructions

Dosage and time

Cell processing and labeling

1

Take the mouse spleen, grind it on a 40 μm sterile filter to prepare a single-cell suspension of the spleen, perform red blood cell lysis, and then count the cells.

Note:It is recommended to count cells after staining with AO/PI; splenic cells 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 reagents proportionally according to the sample volume; set aside a pre-sorting sample to determine the initial B-cell ratio.

1×107cells/100 μL

3

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

Note:In this step, gently pipette up and down 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 minutes;

Incubate at room temperature for 10 minutes

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 minutes;

Incubate at room temperature for 5 minutes

7

Place it in the column selection magnet,L Separation Column, add 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

B cells were enriched from mouse splenocytes using the Mouse Pan B-column-based sorting kit. Cells were stained with CD19-Alexa Fluor® 647 (SDT Cat: S0B5445) and CD3-Alexa Fluor® 488 (SDT Cat: S0B8364) fluorescent antibodies, and analysis was performed by gating on live cells (Live+), allowing for the determination of the proportion of B cells in the sorted fraction. Flow cytometry results indicated that the purity of B cells after this sorting step was 98.8%.