I'm relatively new to flow cytometry work. I harvest mouse samples (blood, lymph node, spleen) and process them for flow analysis:
Single cell suspension --> lyse --> stain --> wash X2 --> flow
Upon flow analysis with FlowJo, I find that when I plot FSC-A by SSC-A there are two distinct densities in the lymph region.
To make sure these populations include lymphocytes I selected from all events CD3+ > CD4+ and overlayed it on the original ungated plot. Both densities show double positive staining indicating to me that they include lymphocytes.
Should I be concerned that my lymph population has 2 distinct size populations? Is there a way to fix this or is it not an issue?
The presence of two distinct populations within the lymph region in your flow cytometry data could be indicative of different subpopulations of lymphocytes. Lymphocytes are a heterogeneous group of immune cells, and different subsets can exhibit variations in size, granularity, and other characteristics.
The two populations you observe could represent different lymphocyte subtypes, such as different stages of activation, maturation, or differentiation. These subpopulations might have distinct functional properties or could be responding differently to the experimental conditions. It's not uncommon to observe such heterogeneity within lymphocyte populations.
To gain more insight into the nature of these populations, it would be beneficial to further characterize them using additional markers specific to different lymphocyte subtypes. By examining the expression of surface markers associated with specific subpopulations (e.g., CD4, CD8, CD19, etc.), you can identify and isolate the different lymphocyte subsets present in your samples.
In terms of fixing this issue, it's important to ensure that your staining and gating strategies are accurate and consistent. Proper compensation and appropriate gating are crucial to accurately identify and analyze specific cell populations. Double-check your staining protocol, antibody panel, and instrument settings to minimize technical artifacts and optimize data quality.
Overall, the presence of two distinct populations within your lymphocyte gate is not necessarily a cause for concern. It may reflect the natural heterogeneity within the lymphocyte compartment. However, further investigation and characterization of these populations using additional markers will provide more information about their identities and potential functional differences.
I only have 1 CD3+ population in flow cytometry. Population 1 could be cell debris. Have you checked viability using a Live/Dead staining? Have you selected only single cells using FSC-A/FSC-H graph?
Moreover, make sue to correctly adjust laser power. For example, you seem to have a lot of cells on the extremity of your FSC scale. If the proper adjustments were made, your CD3+ population 1 would be probably found with the other cell debris.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies