Human CD319 Enrichment Kit

Human CD319 Enrichment Kit

Cat. #: CTG-MGE2738

Kit Components (Contact us for antibody information):

2 ml Human CD319 Magnetic beads conjugate to monoclonal against Human CD319.

100 µl Cell Dissociation reagent (Store at -20^oC)

10 ml Dilution Buffer.

Description

This kit is developed to isolate and enrich the Human CD319+ cells using magnetically labeled with Magnetic beads. The cells are labeled by Human CD319 magnetic beads, the Human CD319⁺ cells are positively selected and released from magnetic beads using our dissociation buffer for further experiment.

Target Features

Symbol: Human CD319

Gene ID: 57823

Uniprot Entry: Q9NQ25

Protein Name: SLAM family member 7 Homo sapiens (Human)

Target Function

Self-ligand receptor of the signaling lymphocytic activation molecule (SLAM) family. SLAM receptors triggered by homo- or heterotypic cell-cell interactions are modulating the activation and differentiation of a wide variety of immune cells and thus are involved in the regulation and interconnection of both innate and adaptive immune response. Activities are controlled by presence or absence of small cytoplasmic adapter proteins, SH2D1A/SAP and/or SH2D1B/EAT-2. Isoform 1 mediates NK cell activation through a SH2D1A-independent extracellular signal-regulated ERK-mediated pathway (PubMed:11698418). Positively regulates NK cell functions by a mechanism dependent on phosphorylated SH2D1B. Downstream signaling implicates PLCG1, PLCG2 and PI3K (PubMed:16339536). In addition to heterotypic NK cells-target cells interactions also homotypic interactions between NK cells may contribute to activation. However, in the absence of SH2D1B, inhibits NK cell function. Acts also inhibitory in T-cells (By similarity). May play a role in lymphocyte adhesion (PubMed:11802771). In LPS-activated monocytes negatively regulates production of proinflammatory cytokines (PubMed:23695528). {ECO:0000250|UniProtKB:Q8BHK6, ECO:0000269|PubMed:11698418, ECO:0000269| PubMed:11802771, ECO:0000269|PubMed:16339536, ECO:0000269|PubMed:23695528, ECO:0000269|Ref.4}.;Isoform 3 does not mediate any NK cell activation.

Capacity        

1 X 10⁹ Cells

Formulation  

All components are supplied in buffer containing stabilizer and 0.05% sodium azide.

Storage & Usage      

Store protected from light at 2−8 °C. Do not freeze. The expiration date is indicated on the vial label. The product is for Research Only, Not for use in Diagnostic Procedures.

General Protocol

The enrichment kit is developed to isolate the target cells with the specific antigen. The procedure is performed by indirectly magnetically labeling the cells with the monoclonal antibody magnetic beads against the specific antigen on the target cell surface, then the labeled cells are subsequently collected over a column, then the target cells are released using our dissociation buffer for future experiment.

1. Sample and reagent preparation

• When working with anticoagulated peripheral blood or buffy coat, peripheral blood mononuclear cells (PBMCs) should be isolated by density gradient centrifugation.
• To remove platelets after density gradient separation, resuspend cell pellet in buffer and centrifuge at 200×g for 10−15 minutes at 20 °C. Carefully aspirate supernatant. Repeat washing step.
• When working with tissues or lysed blood, prepare a single-cell suspension using standard methods.
• Dead cells may bind non-specifically to Magnetic beads. To remove dead cells, we recommend using density gradient centrifugation or the Dead Cell Removal Kit.
• Keep the cells cold, and use pre-cooled solutions.
• Prepare the cell dissociation buffer by mixing the cell dissociation reagent and dilution buffer by 1:100 vol/vol.

2. Magnetic labeling

• Volumes for magnetic labeling given below are for up to 10⁷ total cells. When working with fewer than 10⁷ cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.
• Primary dye-conjugated antibodies should be titrated to determine the optimal staining dilution. Staining should not increase fluorescence intensity of the negative population.

• Calculate the number of cells.
• Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
• Resuspend cells in 80 µL of buffer per 10⁷total cells.
• Add 20 µL of Anti-cell surface antigen Magnetic beads per 10⁷ total cells.
• Mix well and incubate for 15 minutes at 2−8 °C.
• Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 min.
• Aspirate supernatant completely.
• Resuspend up to 10⁸ cells in 500 µL of buffer. Note: For higher cell numbers, scale up buffer volume accordingly.
• Proceed to magnetic separation.

3. Target cell enrichment

• Place column in the magnetic field of a suitable Separator.
• Prepare column by rinsing with the appropriate amount of buffer.
• Apply cell suspension onto the column.
• Collect unlabeled cells that pass through and wash column with the appropriate amount of buffer. Perform washing steps three times.
• Remove column from the separator and place it on a suitable collection tube.
• Pipette 100µ of dissociation buffer onto the column and release the beads into a new eppendorf tube, close and incubate the tube for 15 min at 37^o
• Apply the beads solution to a new magnetic column, collect the released cells in the flow-through fraction which pass through and wash column with 2 × 1 mL of medium. Collect total effluent.
• Harvest the released cells by centrifugation at 300×g for 10 minutes, aspirate the supernatant carefully. Wash the cells with medium twice.
• Resuspend the released cells in medium or desired buffer for further experiment.