Hi al! This morning I run FACs with CHO cells stained with FVD. Unfortunately, the results showed binding with proteins they shouldn't bind with based on bibliography and cell viability was LOW. The protocol and the majority of the materials I used where the same I use for my human cell lines, would this affect the experiment?
Do you have any similar experience?
My materials:
-Cho cells
-human blocking serum
-mouse and rat anti-human IgG
-used efluor 450 but due to low viability I may use PI next time
Saif Wahid
Species-Specific Reagents:Human Blocking Serum: This might not effectively block non-specific binding in CHO cells, leading to increased background and non-specific binding. Mouse and Rat Anti-Human IgG: These antibodies are specific to human proteins and may not bind appropriately or specifically to CHO cell proteins, potentially leading to non-specific staining or lack of signal. Cell Viability:Cell Handling: CHO cells may have different handling and staining requirements compared to human cells. Ensure that the conditions used for staining (e.g., incubation times, temperatures) are suitable for CHO cells. Staining Protocol: Some dyes or reagents may be more toxic to CHO cells than to human cells. Optimizing the staining protocol specifically for CHO cells is essential. Viability Dye:FVD (Fixable Viability Dye): If this dye did not perform well, it might be due to the specific conditions or the inherent sensitivity of CHO cells to the dye. PI (Propidium Iodide): Using PI can help as it is a common viability dye for distinguishing live from dead cells, but it requires careful handling since it only stains dead cells and needs to be added just before analysis. Use Species-Specific Reagents:Blocking Serum: Use blocking serum that is compatible with CHO cells, such as normal hamster serum, to reduce non-specific binding. Antibodies: Ensure that the antibodies used are cross-reactive with hamster proteins or find alternatives specifically validated for use with CHO cells. Optimize Staining Protocol:Incubation Conditions: Verify that the incubation times, temperatures, and buffer conditions are optimal for CHO cells. For example, the cell concentration during staining and the wash steps might need adjustment. Cell Handling: Handle CHO cells gently to maintain viability. Avoid harsh centrifugation speeds and long incubation times in potentially toxic buffers. Alternative Viability Dyes:PI Staining: If you decide to use PI, add it just before FACS analysis to ensure accurate viability assessment. PI is a membrane-impermeable dye that only enters dead cells, making it a reliable indicator of cell viability. Other Viability Dyes: Consider other viability dyes like 7-AAD or Annexin V/PI staining for more comprehensive viability assessment. Controls:Include appropriate controls to differentiate between specific and non-specific binding. Use unstained cells, single-stained controls, and isotype controls to set up proper gating and compensation. Blocking Step:Block with 2% hamster serum or an appropriate species-specific blocking reagent instead of human serum. Incubate the cells with the blocking reagent for at least 15 minutes at 4°C before staining. Staining Step:Adjust antibody concentrations and incubation times specifically for CHO cells. Ensure that all reagents are compatible with CHO cells and validate their performance. Viability Assessment:Use PI (Propidium Iodide) or another appropriate viability dye. Add the dye just before analysis (e.g., 5 minutes before running the samples on the flow cytometer).
Potential Issues:
Recommendations:
Example Protocol Adjustments:
0 votes
0 thanks
More Eleni Kokalopoulou's questions See All
Similar questions and discussions