Rat EPHA5 Bead-based Screening Set
Cat. #: CTG-BSM1376
Kit Components (Contact us for antibody information):
2 ml Rat EPHA5 Magnetic beads conjugate to monoclonal antibody against Rat EPHA5.
100 µl Rat EPHA5 Monoclonal antibody, R-PE conjugated (for detection)
100 µl Cell Dissociation reagent (Store at -20oC)
10 ml Dilution Buffer.
This kit is developed to separate the Rat EPHA5+ cells using magnetically labeled with Magnetic beads for further applications. To deplete and enrich Rat EPHA5 positive cells, the cells mixed with Rat EPHA5 magnetic beads, the Rat EPHA5+ cells are depleted from the samples in the flow-through fraction, or collected and released from magnetic beads using our dissociation buffer for further experiment.
Symbol: Rat EPHA5
Gene ID: 79208
Uniprot Entry: P54757
Protein Name: Ephrin type-A receptor 5
Organism: Rattus norvegicus (rat)
1 X 109 Cells
All components are supplied in buffer containing stabilizer and 0.05% sodium azide.
Storage & Usage
Store protected from light at 2−8 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.
The screening beads are provided to isolate the target cells with mutated cell-surface antigen or to deplete the nontarget cells with the specific antigen. The screening procedure is performed by indirectly magnetically labeling the cells with the monoclonal antibody magnetic beads against the cell-surface antigen (mutated or specific to the nontarget cells), then the labeled cells are subsequently depleted by separation over a Column, and the target cells in the flow-through fraction are collected for further 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 cells in 80 µL of buffer per 107 total cells. Note: Aliquot an appropriate amount of cells for antibody-R-PE labeling and detection.
4) Add 20 µL of Anti-cell surface antigen Magnetic beads per 107 total cells.
5) Mix well and incubate for 15 minutes at 2−8 oC.
6) Wash cells by adding 1–2 mL of buffer per 107 cells and centrifuge at 300×g for 10 min.
7) Aspirate supernatant completely.
8) Resuspend up to 107 cells in 500 µL of buffer. Note: For higher cell numbers, scale up buffer volume accordingly.
9) Proceed to magnetic separation.
3. Depletion of targeted cells
1) Place Column in the magnetic field of a suitable Separator.
2) Prepare column by rinsing with 2 mL of buffer.
3) Apply cell suspension onto the column.
4) Collect unlabeled cells which pass through and wash column with 2 × 1 mL of buffer.
5) Perform washing steps by adding buffer successively once the column reservoir is empty. Collect total effluent. This contains the unlabeled pre-enriched plasma cell fraction.
6) Harvest the target cells in the flow-through fraction by centrifugation at 300×g for 10 minutes, aspirate the supernatant carefully.
7) Resuspend the untouched cells in medium or desired buffer for further experiment.
8) Aliquot an appropriate amount of cells for antibody-R-PE labeling and detection if there is enough cells, otherwise, perform the antibody-R-PE labeling and detection after cell amplification.
4. Target cell enrichment
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 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 37oC.
7) 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.
8) Harvest the released cells by centrifugation at 300×g for 10 minutes, aspirate the supernatant carefully. Wash the cells with medium twice.
9) Resuspend the released cells in medium or desired buffer for further experiment.