Human EPHB3 Bead-based Assay Set For Flow Cytometry

Human EPHB3 Bead-based Assay Set For Flow Cytometry

Cat. #: CTG-BAH1187

Kit Components (Contact us for antibody information):

2 ml monoclonal antibody against Human EPHB3, R-PE Conjugated.

2 ml Anti-R-PE magnetic beads.

1 ml Bead release reagent and 1 ml Stop Buffer

10 ml Dilution Buffer.

Description

This kit is developed to isolate and detect the Human EPHB3⁺ cells using magnetically labeled with Magnetic beads. First, the cells are labeled by Human EPHB3 monoclonal antibody conjugated to R-PE and anti-R-PE magnetic beads, the Human EPHB3⁺ cells are positively selected on a magnetic column, are collected for further flow-cytometric analysis. Bead release reagent and Stop Buffer can be used to release the cell with antibody-R-PE from magnetic beads.

Target Features

Symbol: Human EPHB3

Gene ID: 2049

Uniprot Entry: P54753

Protein Name: Ephrin type-B receptor 3 (EC 2.7.10.1) (EPH-like tyrosine kinase 2) (EPH-like kinase 2) (Embryonic kinase 2) (EK2) (hEK2) (Tyrosine-protein kinase TYRO6)       

Organism:Homo sapiens (Human)

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^oC. 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 detection kit is developed to isolate and detect the target cells with the specific antigen. The screening procedure is performed by directly magnetically labeling the cells with the monoclonal antibody against the specific antigen, conjugated to R-PE and Anti-R-PE magnetic beads, then the labeled cells are subsequently retained to a magnetic column, and the target cells are released and collected for further flow cytometric analysis.

1. Sample 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^oC. 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.

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.

1)    Calculate the number of cells.

2)    Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.

3)    Resuspend cell pellet in 80 µL of buffer per 10⁷ total cells.

4)    Add 20 µl of the primary PE-conjugated antibody and mix well and incubate for 10 minutes in the dark at 2−8 ^oC.

5)    Wash cells to remove unbound primary antibody by adding 1−2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 minutes.

6)    Aspirate supernatant completely and resuspend cells in 80 µL of buffer per 10⁷ total cells.

7)    Add 20 µL of Anti-PE Magnetic beads per 10⁷ total cells.

8)   Mix well and incubate for 15 minutes at 2−8^oC.

9)   Wash cells by adding 1–2 mL of buffer per 10⁷ cells and centrifuge at 300×g for 10 min, aspirate supernatant completely.

10) Resuspend up to 10⁷ cells in 500 µL of buffer. Note: For higher cell numbers, scale up buffer volume accordingly.

11) Proceed to magnetic separation.

3. Positive selection of target cell for detection

1) Place column in the magnetic field of a suitable Separator. 

2) Prepare column by rinsing with the appropriate amount of buffer.

3) Apply cell suspension onto the column.

4) Collect unlabeled cells that pass through and wash column with the appropriate amount of buffer. Perform washing steps three times.          

5) Remove column from the separator and place it on a suitable collection tube.

6) Pipette appropriate amount of buffer onto the column and release the target cells for further flow cytometric analysis.