Mouse Pan B positive selection kits were used to enrich Pan B cells from mouse splenocytes. Cells were stained with CD19-Alexa Fluor® 647 (SDT Cat: S0B5445) and CD3-Alexa Fluor® 488 (SDT Cat: S0B8364) fluorescent antibodies, and live cells (Live+) were gated for analysis to determine the proportion of Pan B cells in the sorted fraction. Flow cytometry results showed that the purity of Pan B cells after this sorting was 98.8%.
Mouse Pan B Cell Isolation Kit (Column-Based)
Mouse Pan B Cell Isolation Kit (Column-Based)
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Product Details
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. |
| 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 | 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 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 defining 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 throughout development and activation. In immunological research, obtaining highly pure, highly active Pan B cells is essential for conducting functional analyses and signaling pathway studies. Unlike conventional mouse B-cell isolation kits, Pan B-cell isolation fully preserves CD43-positive B-cell subsets, including activated B cells, plasma cells, and CD5-positive B-1a cells, thereby comprehensively covering B-cell populations across different developmental stages and 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 operates 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, monocytes, and red blood cells), followed by efficient removal of non-target cells via streptavidin-coated magnetic nanoparticles. Throughout the process, no antigens on the B-cell surface are contacted, maximizing the preservation of cellular morphology, surface marker integrity, and physiological functional activity, thus providing high-quality cell material for downstream experiments.
Protocol
NoteThis kit does not include sorting buffer; please provide it separately. The corresponding Stater product number is:S0D3018.
withL Separation ColumnFor example
Steps |
Operating Instructions |
Dosage and timing |
|
Cell processing and labeling |
1 |
The spleen was harvested from mice, passed through a 40-μm sterile filter for homogenization to prepare a single-cell suspension, and subjected to red blood cell lysis prior to cell counting. 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 a pre-sorting sample for determining the initial B-cell proportion. |
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 pipetted 2-3 times to ensure uniformity. |
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 the Streptavidin Beads thoroughly, then add 10 μL of Streptavidin Beads to 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 the L Separation Column into the column separation magnet and add 3 mL.MagSep Separation Bufferto rinse the separation column. |
Rinse the separation column. |
|
Cell separation. |
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, useMagSep Separation Buffer.Complete; cells that drip down due to gravity are the target cells; |
Collect the target cells |
9 |
Add 3 mLMagSep Separation Buffer, and collect the target cells; Note:This step elutes the unmarked 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 handle a maximum of 1×10⁷ labeled cells.The L Separation Column can handle a maximum of 1×10⁸ labeled cells.; 2.If the viability of the sample is low, it is recommended to use a dead cell removal kit to remove dead cells (Dead Cell Removal Set, (RUO)#S0K0009); | |||
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Validation Data
