CellRapeutics™ NK1.1+ iNKT Cell Isolation Kit (CART-010CL)

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Product Name
CellRapeutics™ NK1.1+ iNKT Cell Isolation Kit

Cat. No.
CART-010CL

Warnings
Reagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with running water before discarding. These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop.

Components
2 mL NK1.1+ iNKT Cell Biotin-Antibody Cocktail, mouse: Cocktail of biotin-conjugated monoclonal anti-mouse antibodies against NKp46, CD45R, CD8a, CD115, and TCRγδ.
1 mL Anti-Biotin MicroBeads: MicroBeads conjugated to monoclonal antibiotin antibody (isotype: mouse IgG1).
1 mL Anti-NK1.1-APC, mouse: Monoclonal anti-mouse NK1.1 antibody conjugated to APC (isotype: mouse IgG2a).
2 mL Anti-APC MicroBeads: MicroBeads conjugated to monoclonal antimouse anti-APC antibody (isotype: mouse
IgG1).

Capacity
For 10⁹ total cells.

Principle of the MACS Separation
The isolation of NK1.1+ iNKT cells is performed in a two-step procedure. First, the non-NK1.1+ iNKT cells are labeled with a cocktail of biotin-conjugated antibodies, Anti-Biotin MicroBeads. The labeled cells are subsequently depleted by separation over a MACS Column, which is placed in the magnetic field of a MACS Separator.
In the second step, the NK1.1+ iNKT cells are labeled with AntiNK1.1-APC and Anti-APC MicroBeads and isolated by positive selection from the pre-enriched cell fraction by separation over a MACS Column, which is placed in the magnetic field of a MACS Separator.
After removing the column from the magnetic field, the NK1.1+ iNKT cells can be eluted as the positively selected cell fraction. To increase the purity, the positively selected cell fraction containing the NK1.1+ iNKTcells must be separated over a second column.

Background Information
Natural killer T (NKT) cells are a heterogeneous group of lymphocytes that share properties of both T cells and natural killer (NK) cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds selfand foreign lipids and glycolipids such as the synthetic ligand α-galactosylceramide (α-GalCer).
The term NKT cells was first used in mice to define a subset of T cells that also express the natural killer cell-associated marker NK1.1 (CD161), although NK1.1 is only expressed by the mouse strain C57BL/6. In general, the term iNKT cells refers preferentially to CD1d-restricted T cells, expressing a heavily biased, semiinvariant T cell receptor (TCR Vα14-Jα18, Vβ8.2, Vβ7 and Vβ2) and NK cell markers.
Upon activation, iNKT cells are able to produce large quantities of IFN-γ, IL-4, and GMCSF, as well as multiple other cytokines and chemokines from Th1- and Th2-type. They are an important link between innate and adaptive immune system, promoting or suppressing immune responses.

Applications
Isolation of of NK1.1-expressing mouse NKT cells after depletion of unwanted cells like NK cells, B cells, makrophages, CD8+, and TCRγδ+ T cells.

Product format/Physical form
All components are supplied in buffer containing stabilizer and 0.05% sodium azide.

Storage Conditions
Store protected from light at 2-8 °C. Do not freeze. The expiration date is indicated on the vial label.

Reagent and instrument requirements
Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS BSA Stock Solution 1:20 with autoMACS Rinsing Solution. Keep buffer cold (2-8 °C). Degas buffer before use, as air bubbles could block the column.
Note: EDTA can be replaced by other supplements such as anticoagulant citrate dextrose formula-A (ACD-A) or citrate phosphate dextrose (CPD). BSA can be replaced by other proteins such as human serum albumin, human serum, or fetal bovine serum (FBS). Buffers or media containing Ca2+ or Mg2+ are not recommended for use. MACS Columns and MACS Separators: Depletion of nontarget cells can be performed on an LD Column. The subsequent positive selection of NK1.1+ iNKT cells can be performed on two MS Columns. Positive selection and depletion can also be performed by using the autoMACS Pro or the autoMACS Separator. Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. (Optional) Fluorochrome-conjugated antibodies for flow cytometric analysis, e.g., CD3ε-VioBlue or Anti-NKp46-FITC.
(Optional) Propidium Iodide Solution or 7-AAD for flow cytometric exclusion of dead cells.
(Optional) Dead Cell Removal Kit for the depletion of dead cells.
(Optional) Pre-Separation Filters (30 µm) to remove cell clumps.

Procedure
Sample preparation When working with lymphoid organs, non-lymphoid tissues, or peripheral blood, prepare a single-cell suspension using manual methods or the gentleMACS™ Dissociator.
▲ Dead cells may bind non-specifically to MACS MicroBeads. To remove dead cells, we recommend using density gradient centrifugation or the Dead Cell Removal Kit.

Magnetic separation: Depletion of non-target cells
▲ Choose an appropriate MACS Column and MACS Separator according to the number of labeled cells and the number of total cells. For details refer to table in section "Reagent and instrument requirements".
▲ Always wait until the column reservoir is empty before proceeding to the next step. 1. Place LD Column in the magnetic field of a suitable MACS Separator. For details see LD Column data sheet.
2. Prepare column by rinsing with 2 mL of buffer.
3. Apply cell suspension onto the column.
4. Collect unlabeled cells that pass through and wash column with 2×1 mL of buffer. Collect total effluent; this is the unlabeled pre-enriched NK1.1+ iNKT cell fraction. Perform washing steps by adding buffer two times. Only add new buffer when the column reservoir is empty.
5. Proceed to section "Magnetic labeling" for the labeling of NK1.1+ iNKT cells.

Magnetic labeling
Depletion with the autoMACS Pro Separator or the autoMACS Separator
▲ Refer to the respective user manual for instructions on how to use the autoMACS Pro Separator or the autoMACS Separator.
▲ Buffers used for operating the autoMACS Pro Separator or the autoMACS Separator should have a temperature of ≥10 °C.
▲ Program choice depends on the isolation strategy, the strength of magnetic labeling, and the frequency of magnetically labeled cells. Magnetic separation with the autoMACS Pro Separator
1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place
sample tube in row A of the tube rack and the fraction collection tubes in rows B and C.
3. For a standard separation choose the following program:
Depletion: deplete05
Collect negative fraction in row B of the tube rack.
4. Proceed to section "Magnetic labeling" for the labeling of NK1.1+ iNKT cells. Magnetic separation with the autoMACS Separator
1. Prepare and prime the instrument.
2. Apply tube containing the sample and provide tubes for collecting the labeled and unlabeled cell fractions. Place
sample tube at the uptake port and the fraction collection tubes at port neg1 and port pos1.
3. For a standard separation choose the following program:
Depletion: deplete05
Collect negative fraction from outlet port neg1.
4. Proceed to section "Magnetic labeling" for the labeling of NK1.1+ iNKT cells.

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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