Hi everyone,
I am a new person currently working on flow cytometry. I got a problem when handling the machine and analyzing my data on Flowjo program.
In my data, the gate of the single cell with FSC-H and FSC-A was shown in the figure below.
I could not figure out what is the problem that causes my data to look awkward as this. (The cells population seems to be cut at the end of the right side of this figure)
Could anyone give me the answer to this situation?
Many thanks!!
Hello,
On the FSC-A FSC-H graph, your cells should be aligned on straight line where x values = y values, much like what you see on the right side of the graph, not like what you have on the left side. Try different combinaison of FSC (A vs H vs W) to gate on a population arranged on a straight line.
I would also recommend trying with Louis-Charles suggested but with SSC
As Louis raise an important point regarding the alignment of cells on the FSC-A FSC-H graph. Ideally, the cells should form a straight line where the x-values (FSC-H) equal the y-values (FSC-A), as observed on the right side of the graph.
By exploring different combinations of forward scatter parameters (FSC-A, FSC-H, and FSC-W), you can experiment with gating strategies that align the cell population along a straight line. This can help in identifying and isolating the desired cell population more accurately
Apart from this, There could be several reasons for this issue. Here are a few possibilities to consider:
1. Instrument settings: Ensure that the flow cytometer is properly calibrated and the instrument settings, including laser power, voltage, and gain, are optimized for your sample. Inaccurate settings can affect the detection and resolution of cell populations.
2. Cell concentration: It is crucial to maintain an appropriate cell concentration within the optimal range for accurate analysis. Very high or very low cell concentrations can impact the data quality and result in distorted populations.
3. Compensation: Check if compensation has been properly applied to correct for spectral overlap between fluorochromes. Incorrect compensation can lead to misinterpretation of the data and affect the placement of gates.
4. Gating strategy: Review your gating strategy to ensure that it accurately defines the desired cell population. Double-check that the gating parameters and thresholds are set appropriately for your specific experiment.
5. Data analysis: When analyzing your data in the FlowJo program, ensure that you have correctly applied the appropriate compensation and gating settings. Incorrect analysis settings or improper application of gates can result in misleading representations of the data.
Dear all,
Thank you for your very helpful suggestions as well as the answers.
I have checked the data again, and now I can figure it out.
Once again, Thank you a lot for your generosity and response.
Best regards,
Trinh
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