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| Product Name | HEK293T-DPP4 Overexpression Cell Line |
| Catalog No. | RESK-0454 |
| Description | Coronaviruses are a class of positive-sense RNA viruses. The spike (S) glycoprotein on the viral envelope mediates receptor recognition through its receptor-binding domain (RBD), and membrane fusion occurs after the S protein is cleaved into S1 and S2 subunits. The RBD of the MERS-CoV spike protein specifically recognizes and binds the cell surface receptor dipeptidyl peptidase 4 (DPP4), mediating viral entry and infection. The RBD is also an epitope-enriched region for MERS-CoV neutralizing antibodies; RBD-targeting neutralizing antibodies can specifically block MERS-CoV infection of DPP4-expressing cells as well as the binding of the RBD to DPP4-expressing cells. This cell line is a stable clone generated by infecting HEK293T cells with a DPP4-overexpression lentivirus followed by selection. |
| Intended Use | For virus infection research, e.g., studies on the invasion mechanism of MERS-CoV. For neutralizing antibody detection, e.g., evaluation of neutralizing antibody activity against the RBD. For research use only. |
| Principle / Technology | Lentiviral transduction. HEK293T cells were infected with a DPP4-overexpression lentivirus, and a stable cell line was obtained after selection with puromycin. The overexpressed DPP4 on the cell surface serves as the receptor specifically recognized and bound by the MERS-CoV spike protein RBD. |
| Detection Method | Trypan blue staining and counting |
| Sample Type | Cell line |
| Performance Range / Specifications | Cell product type: overexpression cell line Cell type: HEK293T Target gene: DPP4 Selection marker: Puromycin Construction method: lentiviral infection followed by stable clone selection Expression type: stable expression Concentration: 5E6 cells/mL Passage ratio: 1:4-1:5 Digestion condition: 0.25% Trypsin-EDTA, 30-60 s Centrifugation condition: 1000 rpm, 3-5 min Cell density: 20-30% after passaging; do not exceed 80% |
| Specificity | The MERS-CoV spike protein RBD specifically recognizes and binds the cell surface receptor DPP4 overexpressed on this cell line. |
| Reaction Conditions / Protocol | Cell Revival: 1) Pre-warm the medium in a 37 °C water bath; add 5 mL of pre-warmed complete medium to a 15 mL centrifuge tube. 2) Remove the frozen cells (5E6 cells/mL) from liquid nitrogen and immediately place the vial in a 37 °C water bath; immerse the vial below the water surface and shake gently until thawed. 3) Wipe the outside of the cryovial with 70% ethanol to reduce the risk of contamination. 4) In a biosafety cabinet, transfer the cell suspension to the prepared 15 mL centrifuge tube, mix gently, and centrifuge at 1000 rpm for 5 min; discard the supernatant. 5) Resuspend the cells in 1 mL of medium; the viable cell density should be 3.5E6-5E6 cells/mL. A portion of the cells may be taken for viable cell counting by trypan blue staining. 6) Seed the cells into one 10 cm dish (10 mL, 78 cm2 growth area) and one 6 cm dish (5 mL, 28 cm2 growth area) at a density of 20-30%. 7) Incubate in a 37 °C incubator for 24 h and examine cell attachment under a microscope. Once attached, carefully replace the medium or passage the cells according to cell density. Passage the cells when confluence exceeds 60%; the density must not exceed 80%. If attachment is incomplete, continue incubation up to 48 h. 8) The first passage can usually be performed about 48 h after revival. Cell Passaging: 1) Digest the cells with 0.25% Trypsin-EDTA for 30-60 s. 2) Passage adherent cells according to confluence at a recommended split ratio of 1:4-1:5, every other day. Note: passage when confluence is above 60%; the density must not exceed 80%. 3) Aspirate the culture medium from the dish or flask and rinse the cells with an appropriate volume of PBS 1-3 times. 4) Discard the PBS, add 1 mL of cell dissociation solution, and digest in a 37 °C incubator for 30-60 s; observe under the microscope until the cells become rounded, intercellular gaps are apparent, and some cells begin to detach from the vessel wall. 5) Add about 2 mL of complete medium to stop the digestion, gently pipette the cells off, and centrifuge at 1000 rpm for 3 min at room temperature. 6) Discard the supernatant, resuspend the cell pellet in complete medium, and plate the cells according to the pre-passage density (calculated from the dish area and cell density) so that the post-passage density is 20-30%. Cell Cryopreservation: 1) Prepare 2x cell freezing medium: 80% FBS + 20% DMSO. 2) Count the cells and collect them by centrifugation at 1000 rpm for 3 min. 3) Adjust the cell density to 2x with pre-cooled FBS. 4) Add an equal volume of 2x cell freezing medium and mix gently. 5) Dispense 1 mL per cryovial; the freezing volume is 1 mL and the freezing density is 5E6 cells/mL. 6) After labeling, place the cryovials in a controlled-rate freezing container at -80 °C for at least 1 day, and transfer to liquid nitrogen as soon as possible. |
| Components / Formulation | Recommended growth medium: 90% DMEM + 10% FBS + 1% P.S + 0.75 μg/mL Puromycin Recommended freezing medium: 90% FBS + 10% DMSO |
| Storage Conditions | Store in liquid nitrogen. If the cells are not recovered immediately upon receipt, transfer them to liquid nitrogen storage at once. |
| Package Specifications | 1 mL |
| Product Form | Frozen cell suspension, 5E6 cells/mL |
| Quality Control | After revival, the viable cell density should be 3.5E6-5E6 cells/mL as determined by trypan blue staining and counting. |
| Concentration | 5E6 cells/mL |
| Source / Origin | HEK293T cells (human origin) transduced with a DPP4-overexpression lentivirus and selected as a stable cell line. |
| Regulatory / Compliance | For research use only. |
| Recommended Buffer System | Growth medium: 90% DMEM + 10% FBS + 1% P.S + 0.75 μg/mL Puromycin Freezing medium: 90% FBS + 10% DMSO Dissociation reagent: 0.25% Trypsin-EDTA Wash buffer: PBS |
| Application Notes / Precautions | Wipe the outside of the cryovial with 70% ethanol before opening to reduce the risk of contamination. Passage the cells when confluence exceeds 60%; the cell density must not exceed 80%. After the first revival, the first passage can usually be performed at about 48 h. Maintain puromycin selection (0.75 μg/mL) in the growth medium to preserve stable DPP4 expression. |
For research use only, not for clinical use.
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