Hello Scientific World,
I would highly appreciate some help here. I am using THP-1 monocytes as my pet cell line to generate human macrophages. Before every differentiation process, I test my THP-1 cells for correct marker decoration on the cell surface by flow cytometry.
Marker presentation is hardly a problem as I get CD14 and CD11b markers detected along with some others that I look for.
Problem:
However, I get different population at the different positions in the scatter plot (FSC-H and SSC-H). Please look at the picture attached. The heat map shows show dense population and its tricky for me to gate. I have to usually separate these population for analysis since they influence autofluorescence in the FITC channel.
Please suggest,
Thanks,
Sudip
Hello
i think at first you have to see which population you need. here its like too much noisy and with debris cells. I think you should do Dot plot and Histogram one time and see the results. Secondly when you selecting the population at first do select in batches and see the results.
With regards.
First i need to suggest on the gating of your cells. I afraid you gated on the monocytes because from the figure it is hard to differentiate the distribution of the cells. can you show us normal cell distribution without smoothing on the flow jo as it makes the cells aggregate to one site.
second, unless you use a dead cell markers, you cant get identify your live population as there are many events during the process making your cells dead.
Follow the following steps to get the specific population or THP-1 monocytes positive
1. gate on monocytes or lymphocytes( FSA vs SSA)
2. select single cells(SSH vs SSA)
3. use viability dye such as FVD and exclude your dead cells if they are required in the assay
4. then identify positive moniocytes which are positive for your marker
cheers and good luck
See my attached picture for my interpretation (based on a 5 or 6 years working with THP-1 cells). You could add various other markers or viability dyes to confirm but it may not be worth doing so depending on your application/experiment.
Presumably you are using PMA to differentiate the cells; it's not unusual to see a reasonable amount of cell death from the PMA treatment, try to wash off the dead cells before you detach and analyse your differentiated THP-1 cells.
I may not be able to answer the question. However, there are several THP-1 cell lines, and one may be contaminated with mycoplasma. Please check the contamination.
I am working with myeloma cells, which are very similar to THP-1, although I agree with other, I would guess that the population are cells which are death, or undergoing apoptosis. Check your cell culture setup and do ficoll. Than try again and the population should disappear.
Wish you best
Hello Marc, thank you for your help. I wanted to let you know that I am using M-CSF to differentiate macrophages. Since we are working with the same system as this paper from our collaborative group: Article Airway Microbiota Determines Innate Cell Inflammatory or Tis...
And I have used PI staining with no PI positive cells (flow plot attached). Although, I did not check for apoptotic cells, I will do it soon. To my surprise after differentiation the corresponding cells in the gate with 85% of cells vanishes after 7 days of differentiation and all cells go to the smaller population (please look at the attached) I have to hunt down the population to low FSC/SSC and then gate, but still dead cells.
Please suggest,
Sudip
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology