Cat.No. : CSC-RO0671
| Cat. No. | CSC-RO0671 |
| Description | This cell line is engineered to stably co-overexpress human PDCD1 and CD81-GFP in a human CD274 knockout cell line. |
| Gene | PDCD1/CD81-GFP |
| Host Cell Species | Homo sapiens (Human) |
| Stability | Validated for at least 10 passages |
| Application | 1. Studying the interactions between immune cells and cancer cells 2. Studying the mechanisms of resistance to immune checkpoint blockade 3. High-throughput screening 4. Drug target validation |
| Quality Control | Negative for bacteria, yeast, fungi and mycoplasma. |
| Shipping | Dry ice |
| Storage | Liquid nitrogen |
| Revival | Rapidly thaw cells in a 37°C water bath. Transfer contents into a tube containing pre-warmed media. Centrifuge cells and seed into a 25 cm2 flask containing pre-warmed media. |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
A: For quantitative analysis of GFP signal intensity in the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line, flow cytometry is highly recommended. This technique allows for the precise measurement of GFP fluorescence, thereby assessing the co-expression levels of CD274(-), PDCD1, and CD81 markers. Before proceeding, cells should be properly prepared and resuspended in a suitable buffer. Employing a flow cytometer calibrated for GFP detection ensures accurate measurement. Additionally, data analysis software can be used to analyze fluorescence intensity, providing insights into the expression levels of these proteins. To complement flow cytometry, fluorescence microscopy can be employed to visualize the distribution and localization of GFP-tagged proteins within the cells.
A: Enhancing transfection efficiency in the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line, especially for studies focusing on gene overexpression or knockdown, requires optimization of several key parameters. Electroporation or lipid-based transfection methods should be carefully selected based on their compatibility with this cell line. Optimization involves adjusting the DNA or siRNA concentration, the volume of transfection reagent, and the ratio of DNA to reagent. Additionally, performing a pilot study to determine the optimal conditions for transfection without affecting cell viability is crucial. Post-transfection, the efficiency can be evaluated using quantitative PCR or Western blot for the target gene, ensuring that the modifications do not interfere with the baseline GFP expression or the cell line's stability.
A: When designing drug screening assays with the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line to identify inhibitors of the PD-1/PD-L1 pathway, it's essential to consider several critical factors. First, the choice of assay format (e.g., high-throughput screening compatible) and detection method (e.g., fluorescence-based readouts) should align with the GFP expression. It's vital to establish baseline GFP fluorescence intensity to accurately measure any modulation upon drug treatment. Furthermore, including a range of drug concentrations to determine the dose-response curve is crucial for assessing drug efficacy. Control treatments, such as known PD-1/PD-L1 pathway inhibitors, should be included to validate the assay's sensitivity and specificity. Lastly, evaluating cell viability alongside the primary assay endpoints is necessary to distinguish between specific pathway inhibition and general cytotoxic effects.
A: To study the molecular interactions between PD-1 and its ligands using the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line, co-immunoprecipitation (Co-IP) followed by Western blot analysis is highly effective. This approach allows for the detection of physical interactions between PD-1 and its ligands by immunoprecipitating PD-1 or its ligands from the cell lysates and probing with specific antibodies against the interaction partners. Additionally, fluorescence resonance energy transfer (FRET) can be utilized to study the proximity between PD-1 and its ligands in live cells, with GFP serving as a fluorescent reporter. These experiments can be complemented with functional assays, such as T-cell activation or inhibition assays, to understand the biological significance of these interactions.
A: Ensuring the maintenance of GFP fluorescence and stability of transgene expression in the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line during long-term culture requires meticulous cell culture practices. This includes using a consistent culture medium and conditions recommended by the cell line provider, regularly monitoring GFP expression levels through flow cytometry or fluorescence microscopy, and avoiding over-confluency by subculturing cells at appropriate intervals. It's also advisable to freeze down multiple aliquots of cells at early passages to preserve the original expression characteristics. Periodic validation of transgene expression through molecular techniques such as qPCR or Western blotting for GFP and the target transgenes is recommended to confirm stability over time.
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This cell line uniquely incorporates GFP (green fluorescent protein), which is fused to the CD81 protein, allowing for clear visualization under fluorescent microscopy. The Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line enables us to observe CD81 localization and trafficking in real time, providing insights into cellular dynamics and interactions.
The Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line has both CD274 and PDCD1 knocked out, which allows for the study of immune checkpoint pathways without the interference of these proteins. This dual knockout feature is invaluable for us focusing on the independent roles of other immunological markers like CD81 in cellular processes.
By lacking CD274 and PDCD1, this cell line serves as an excellent control for studying the specific effects of therapies targeting these pathways. The Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line provides a clear baseline to assess the impact of manipulating other molecular targets in the context of immune modulation.
The specific configuration of this cell line—combining knockouts with a fluorescent marker—enables complex studies of immune interactions and functions. We can use the Human CD274(-)/PDCD1/CD81-GFP Stable Cell Line to dissect the multifaceted roles of CD81 in the immune system, particularly in the presence or absence of typical checkpoint inhibitions.
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