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Mouse Lineage Cell Depletion Kit (Column-Free)

Mouse Lineage Cell Depletion Kit (Column-Free)

Catalog Number: S0K3018 Reactivity: Human Conjugation: Brand: Starter
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Product Details

Product Specification


Format

1 mL Mouse Lineage Cell Depletion Biotin-Antibody Cocktail

1 mL Streptavidin NanoBeads
Capacity

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

This kit employs a column-free negative selection method to isolate lineage-negative (Lin⁻) stem cells/progenitor cells from mouse bone marrow. In this negative selection process, mature lineage cells are labeled with biotin-conjugated antibodies, and the cells are then incubated with streptavidin-nanomagnetic beads. Non-target cells are labeled via antibody-bead complexes, while the target lineage-negative (Lin⁻) stem cell/progenitor cell population remains 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 lineage-negative (Lin⁻) stem cells/progenitor cells remain in the supernatant; this supernatant consists of untouched cells that have not been labeled with magnetic beads and retain their original cellular activity.
Applications in cell sorting Label-free, native lineage-negative (Lin⁻) stem/progenitor cells are sorted from fresh mouse bone marrow single-cell suspensions and can be 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

Lin⁻ cells are a population of primitive cells in mice that do not express any surface lineage markers characteristic of mature, terminally differentiated cells. They are predominantly found in bone marrow tissue, with smaller numbers present in hematopoietic tissues such as the spleen. This cell population primarily comprises hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HSPCs), representing undifferentiated cells within the hematopoietic system that possess self-renewal capacity and multipotential differentiation potential, distinct from fully committed, functionally specialized mature hematopoietic cells. The purified primitive cell population obtained by depleting all mature hematopoietic lineages—such as T cells, B cells, monocytes/macrophages, granulocytes, and erythroid cells—serves as the foundational enriched fraction for isolating and studying hematopoietic stem and progenitor cells. Using immunomagnetic negative selection, a mixture of biotinylated lineage antibodies specifically labels all Lin⁺ mature hematopoietic cells, which are then selectively bound by streptavidin-coated magnetic beads. These labeled cells are retained via magnetic field adsorption, while the unlabeled Lin⁻ primitive stem and progenitor cells, unaffected by the magnetic field, pass through and are collected. A key advantage of this negative sorting technique is its contact-free nature: target cells remain unexposed to antibodies and magnetic beads, thereby avoiding surface antigen cross-linking, cell activation, or damage, thus preserving stem cell stemness, viability, and differentiation potential to the greatest extent—superior to positive selection—and making it well-suited for demanding stem cell functional assays.

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

Collect mouse bone marrow, prepare a single-cell suspension, and perform cell counting;

Note:It is recommended to count cells after staining with AO/PI;

Prepare the sample

2

Resuspend 1×10⁷cells in 100 μL of pre-cooledMagSep Separation Buffer(Cat:S0D3018);

Note:Adjust reagent volumes proportionally based on sample volume; set aside a pre-sorting sample to determine the Lin- cell percentage;

1×10⁷cells/100 μL

3

Add 10 μL of Mouse Lineage Cell Depletion Biotin-Antibody Cocktail to the sample and gently mix;

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

10 μL/100 μL

Incubate the antibodies and cells in the cocktail at 2-8℃ for 10 min;

Incubate at 2-8℃ for 10 min

5

Vortex for 5-30 s to mix the Streptavidin NanoBeads thoroughly;

Mix for 5-30 s

6

Add 10 μL of Streptavidin NanoBeads to the sample;

10 μL/100 μL

7

Gently mix the magnetic beads and cells, then incubate at 2-8℃ for 5 min;

Note:In this step, mixing can be done by gently pipetting up and down 2-3 times;

During this step, the magnetic beads may settle at the bottom; you can gently tap the tube twice to remix them;

Incubate at 2-8℃ for 5 min;

8

Add pre-cooledMagSep Separation Bufferto the sample, and immediately place it on the magnetic stand to prevent the beads from settling heavily;

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

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

9

The sample is placed on the magnetic pole, causing the magnetic beads to adhere to the tube wall.

Single-well magnetic rack adsorption (5 min)

Adsorption on a magnetic rack (8 min)

10

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 400g for 7 minutes and used for subsequent culture and analysis.

Cell sorting successful.

Note:1. 0.1-1 × 10⁸Cells (0.1–1 mL) are recommended to be used with a 5 mL single-well magnetic stirrer bar.Starter EasyIso Separator) The total adsorption volume is 2.5 mL;

2-5 ×10⁸For cells (2–5 mL), it is recommended to use a 15 mL multi-position magnetic stirrer.Starter EasyEights EasyIso Separator) The total adsorption volume is 7.5 mL;

2. Throughout the entire process, pay close attention to temperature control; keep the sorting buffer pre-cooled at all times and avoid leaving it at room temperature for extended periods.


Picture

Validation Data

Lineage-negative (Lin⁻) stem/progenitor cells were enriched from mouse bone marrow cells. Cells were stained with the Lineage Cocktail-BV421 fluorescent antibodies (anti-mouse CD3, clone 17A2; anti-mouse Ly-6G/Ly-6C, clone RB6-8C5; anti-mouse CD11b, clone M1/70; anti-mouse CD45R/B220, clone RA3-6B2; anti-mouse TER-119/erythroid cells, clone Ter-119), and live cells (Live⁺) were gated for analysis to determine the proportion of Lin⁻ stem/progenitor cells in the sorted fraction. Flow cytometry results indicated a purity of 88.7% for this sorting.