What to do first flow cytometry

Flow cytometry is a powerful technique used to analyze and sort cells based on their physical and chemical characteristics. When setting up a flow cytometry experiment, there are several important steps to consider. Here is a general flowchart of what to do first in a flow cytometry experiment:

  1. Define Your Experimental Objectives:
  • Clearly define the research questions or objectives of your experiment. Understand the specific information you want to gain from the analysis.
  1. Select Fluorochromes and Antibodies:
  • Choose fluorochromes and antibodies that are specific for the cellular markers or proteins of interest. Consider the fluorochrome compatibility with your flow cytometer’s laser and filter configurations.
  1. Prepare and Label Cells:
  • Harvest and prepare your cells for analysis. Label the cells with fluorochrome-conjugated antibodies. Include appropriate controls, such as unstained cells, single-stained controls, and isotype controls.
  1. Stain Viability Marker:
  • Include a viability marker (e.g., propidium iodide, 7-AAD) to discriminate between live and dead cells. This step helps in excluding non-viable cells from the analysis.
  1. Fixation/Permeabilization (if required):
  • Depending on your experimental setup, you may need to fix and permeabilize cells to allow intracellular staining. Follow the recommended protocols for fixation and permeabilization for your specific cell type and antibodies.
  1. Run Compensation Controls:
  • Set up compensation controls to correct for spectral overlap between fluorochromes. Use single-stained controls for each fluorochrome to create compensation matrices.
  1. Set Up Instrument Parameters:
  • Set up the flow cytometer parameters, such as voltage, threshold, and acquisition settings. Optimize these parameters using single-stained controls to achieve the best resolution between positive and negative populations.
  1. Acquire Data:
  • Acquire data by running your samples through the flow cytometer. Collect sufficient events to ensure statistical significance. Record data on multiple channels to capture information from all fluorochromes.
  1. Data Analysis:
  • Analyze the acquired data using flow cytometry analysis software. Gate populations based on forward scatter (FSC) and side scatter (SSC) to identify cell populations of interest. Perform additional gating based on fluorescent markers.
  1. Interpret Results:
  • Interpret your results based on the identified cell populations and the expression levels of the markers of interest. Draw conclusions related to your research objectives.
  1. Troubleshoot and Optimize:
  • If necessary, troubleshoot any issues that may arise during the analysis. Optimize experimental conditions for future experiments based on the results obtained.

Remember to follow the specific protocols recommended by the manufacturer of your flow cytometer and reagents. It’s also crucial to run appropriate controls for each experiment to ensure the accuracy and reliability of your flow cytometry data.