1 1

Mouse Pan B Cell Isolation Kit (Column-Free)

Mouse Pan B Cell Isolation Kit (Column-Free)

Catalog Number: S0K3007 Reactivity: Mouse Conjugation: Brand: Starter
Share:
Price:
$917 USD
$917 USD
Size:

For shipping services or bulk orders, you may request a quotation.
Secure checkout with
View full details

Product Details

Product Specification


Format

1 mL Mouse Pan B 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

In this negative selection process, non-target cells are labeled with biotin-conjugated antibodies, and then the cells are incubated with streptavidin-nanomagnetic beads. As a result, non-target cells are marked by the antibody‑bead complex, while the targetBcells 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 unlabeledBcells remain in the supernatant. This supernatant constitutes theBcell-enriched fraction after removal of the non-target cells.
Applications in cell sorting High-purity mouse pan-B cells, including conventional B-2 B cells, B-1 B cells, and plasma cells, can be isolated from splenic single-cell suspensions and used for a variety of downstream functional assays.
Reactivity Mouse
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

Mouse Pan B cells (pan-B cells) refer to the entire population of B cells in mice, encompassing all lineages, developmental stages, and functional subtypes. Pan B cells include all B-lineage cells, from early bone marrow progenitor B cells and transitional B cells to peripheral mature follicular B cells (FO B), marginal zone B cells (MZ B), B1 innate-like B cells, memory B cells, and even plasma cell precursors. Their core characteristic is the expression of broad B-lineage hallmark molecules; the classic markers for mouse Pan B cells are CD19 and B220 (CD45R), with CD19 serving as a lineage-specific, invariant marker that remains expressed continuously from early development through the activation phase. In immunological research, obtaining highly pure, highly active Pan B cells is a prerequisite for conducting functional analyses and signaling pathway studies. Unlike conventional mouse B-cell isolation kits, Pan B-cell isolation can fully preserve CD43-positive B-cell subsets, including activated B cells, plasma cells, and CD5-positive B-1a cells, thereby comprehensively covering B-cell populations at various developmental stages and with different functional phenotypes, preventing the loss of critical B-cell subsets and more accurately reflecting the true composition of B cells in vivo. This negative-selection kit is based on the principle of negative selection, using a biotin-labeled antibody cocktail (excluding anti-CD19 antibodies) to specifically label non-B cells (such as T cells, NK cells, monocytes, and red blood cells), followed by efficient removal of non-target cells using streptavidin-coated magnetic nanoparticles. Throughout the process, no antigens on the B-cell surface are directly targeted, which maximizes the preservation of the cells' native morphology, integrity of surface markers, and physiological functional activity, providing high-quality cellular material for downstream experiments.

Protocol

NoteThis kit does not include sorting buffer; please provide it separately. The corresponding Stater product number is:S0D3018.

Steps

Operating Instructions

Dosage and timing

1

The spleen was harvested from mice, passed through a 40-μm sterile filter for homogenization, and a single-cell suspension of splenic cells was prepared; cell counting was then performed.

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

Use 100 μL.MagSep Separation BufferResuspend to 1 × 10⁷cell;

Note:Reagents are adjusted proportionally based on the sample volume; retain the initial mouse B proportion for pre-sorting sample testing.

1 × 10⁷cells/100 μL

3

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

Note:In this step, the mixture can be gently mixed by pipetting up and down 2–3 times.

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

Shake for 5–30 s to mix the Streptavidin beads thoroughly;

Mix for 5–30 s

6

Add 10 μL of Streptavidin beads to the sample;

10 μL/100 μL

7

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

Note:For this step, gently pipette up and down 2–3 times to mix;

During this step, the magnetic beads may settle at the bottom; you can gently flick the tube to remix them after about 2.5 minutes;

Incubate at room temperature for 5 min

8

AddMagSep Separation Bufferto the sample, and immediately place it on a magnetic stand to prevent the beads from settling heavily at the bottom;

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

Add the total volume to 7.5 mL (using a 15 mL centrifuge tube).

9

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

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

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

10

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

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

Cell sorting successful.

Note: 1.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;

For 2–5 × 10^8 cells (2–5 mL), it is recommended to use a 15 mL multi-well magnetic stand (Starter EasyEights EasyIso Separator) with a total adsorption volume of 7.5 mL;

2.If the live cell rate in the sample 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 non-column 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 98.4%.