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Human Pan B Cell Isolation Kit (Column-Free)

Human Pan B Cell Isolation Kit (Column-Free)

Catalog Number: S0K3017 Reactivity: Human Conjugation: Brand: Starter
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$1,170 USD
$1,170 USD
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Product Details

Product Specification


Format

1 mL Human Pan B Cell Biotin Antibody Cocktail;

1.25 mL Streptavidin Beads;

All components are stored in a buffer containing 0.05% sodium azide.
Capacity

Suitable for a total cell count of 1×10⁹, allowing up to 100 sorting runs.
Principle of Separation

In this negative selection process, non-human Pan B cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-coated nanomagnetic beads. As a result, the non-target cells are labeled via the antibody–bead complex, while the target human Pan B cells 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 unlabeled human Pan B cells remain in the supernatant. This supernatant constitutes the enriched fraction of human Pan B cells after removal of the non-target cells.
Applications in cell sorting Pan B cells in their native, unmanipulated state can be isolated from human PBMCs and used for a variety of downstream functional assays.
Reactivity Human
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

Human Pan B cells are a core cell population in the human humoral immune response. They can differentiate into specific antibodies and mediate the formation of immunological memory, serving as an important component of the body's adaptive immune system. They are extensively involved in immune defense, immune regulation, and the development of various immune-related diseases. The Human Pan B Cell Negative Selection Kit is developed based on negative selection technology using immunomagnetic beads, which avoids the antibody-binding labeling defects inherent in positive selection. It can specifically remove various non-target cells, including T cells, NK cells, monocytes, granulocytes, platelets, and red blood cells. The antibody components of this separation system do not contain CD43, effectively preventing the loss of CD43-positive B cell subsets and fully preserving diverse B cell lineages such as naïve B cells, memory B cells, and plasma cells. The resulting high-purity Pan B cells are free from exogenous labels and remain in their native state, maximizing the preservation of their biological potential for proliferation, differentiation, and antibody secretion. This makes them highly suitable for various in vitro functional assays and represents the mainstream tool for isolating and purifying human Pan B cells in the research field today.

Protocol

Note: This kit does not include the sorting buffer; it must be provided separately. The corresponding Stater product number is S0D3018.

Steps

Operating instructions

Dosage and time

1

Isolate PBMCs from peripheral blood using density gradient centrifugation with lymphocyte separation medium, or directly thaw cryopreserved PBMCs, and 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 1×10⁷cells in 100 μL of MagSep Separation Buffer;

Note:Adjust reagent volumes proportionally based on sample volume; retain a pre-sorting sample to determine the proportion of Human Pan B cells;

1×10⁷cells/100 μL

3

Add 10 μL of Human 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 thorough mixing;

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

After antibody incubation, add twice the volume ofMagSep Separation Buffer, centrifuge at 400 g for 7 min, then carefully discard the supernatant. Add 100 μLMagSep Separation BufferResuspend 1×10⁷cells and proceed to the next steps;

Add twice the volume of buffer to wash the antibodies

6

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

Mix for 5–30 seconds

7

Add 12.5 μL of Streptavidin beads to the sample;

12.5 μL/100 μL

8

Gently mix the magnetic beads and cells, and incubate at room temperature for 8 minutes;

Note:In this step, gently pipette up and down 2-3 times to mix thoroughly.

In this step, the magnetic beads may settle at the bottom; you can mix them again after 4 minutes.

Incubate at room temperature for 8 minutes.

9

Add MagSep Separation Buffer to the sample and immediately place it on the magnetic stand to prevent the magnetic beads from settling excessively.

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

Adjust the total volume to 7.5 mL (in a 15 mL centrifuge tube).

10

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

Single-well magnetic rack adsorption (4 min).

Multi-well magnetic rack adsorption (5 min).

11

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

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

Cell sorting successful.

Note:1. 0.1-1×10⁸For 0.1–1 mL of cells, it is recommended to use a 5 mL single-well magnetic separator (Starter EasyIso Separator) with a total adsorption volume of 2.5 mL.

2–5 × 10⁸For 2–5 mL of cells, it is recommended to use a 15 mL multi-well magnetic separator (Starter EasyEights EasyIso Separator) with a total adsorption volume of 7.5 mL.

2. If the sample viability is low, it is recommended to use the Dead Cell Removal Kit (RUO #S0K0009) to remove dead cells.



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Validation Data

Human Pan B cells were enriched from peripheral blood mononuclear cells (PBMCs). After sorting, the cells were stained with CD19-APC (Clone: HIB19) and CD43-PE (Clone: CD43-10G7) fluorescent antibodies, and analyzed by gating on live cells (Live⁺) to determine the proportion of Pan B cells in the sorted fraction. Flow cytometry results showed that the purity of CD19⁺ cells (including both CD43⁺ and CD43⁻) after sorting was 95.85%.