Human EPHA5 Bead-based Assay Set For Flow Cytometry
Cat. #: CTG-BAH1182
Kit Components (Contact us for antibody information):
2 ml monoclonal antibody against Human EPHA5, R-PE Conjugated.
2 ml Anti-R-PE magnetic beads.
1 ml Bead release reagent and 1 ml Stop Buffer
10 ml Dilution Buffer.
This kit is developed to isolate and detect the Human EPHA5+ cells using magnetically labeled with Magnetic beads. First, the cells are labeled by Human EPHA5 monoclonal antibody conjugated to R-PE and anti-R-PE magnetic beads, the Human EPHA5+ 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.
Symbol: Human EPHA5
Gene ID: 2044
Uniprot Entry: P54756
Protein Name: Ephrin type-A receptor 5
Organism:Homo sapiens (Human)
1 X 109 Cells
All components are supplied in buffer containing stabilizer and 0.05% sodium azide.
Storage & Usage
Store protected from light at 2−8oC. Do not freeze. The expiration date is indicated on the vial label. The product is for Research Only, Not for use in Diagnostic Procedures.
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 20oC. 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 2−8oC.
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.