Its an In Vitro Cell Proliferation using Leukemia patient cells lines. Cell Proliferation Dye Used : Tag-It Violet Cell Proliferation Dye. Thank you a lot.
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Gating: As before, exclude debris, doublets, and dead cells based on FSC and SSC. For leukemia cells, you might also consider excluding dead cells using viability dyes like propidium iodide (PI). Find the Proliferation Platform: Navigate to the "Biology" tab and select the "Proliferation" platform. Identify Resting Cells: Since Tag-It Violet is not generation-specific, there won't be distinct peaks for each generation. Instead, look for a broader peak representing resting cells with low dye intensity. Set Gate for Resting Cells: Draw a gate around the peak representing the population of resting cells (low Tag-It Violet intensity). Analyze Proliferating Cells: FlowJo will calculate the percentage of cells with higher Tag-It Violet intensity, which corresponds to proliferating cells. View Results: FlowJo will display the distribution of cells based on Tag-It Violet intensity. You'll get statistics like the percentage of resting cells (gated population) and the percentage of proliferating cells (high-intensity population).
Preprocessing:
Proliferation Analysis:
Output and Statistics:
Additional Considerations:
- Compensation: Ensure compensation is set for Tag-It Violet (usually excited by a violet laser) to avoid overlap with other fluorochromes used for viability staining.
- Positive Controls: Include a positive control, such as stimulated healthy cells, to validate the assay's ability to detect proliferation.
- Cell Line Specificity: Proliferation patterns might vary depending on the specific leukemia cell line used. Consider historical data or pilot experiments to establish baselines.
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