Service – Practical Training

Curriculum: Basic Flow Cytometry Training

Target audience: From complete novice to users how have some knowledge what “FACS” is

Group size: 2 persons should be ideal. Useful to group people according to the experiments they want to perform. For example, cell cycle, reporter protein detection, functional assay, Immunophenotyping or any other assay if user is willing to provide reagents.

Time: 4 full days

Method: I will use white board and PowerPoint; this is highly interactive. Last 2 days will be hands-on training. We will evaluate the learning quality the following week using an experiment done by the user independently, by assessing the data.

Goal: This course is designed to make users aware of what Flow Cytometry can offer, NOT to train someone to become a Flow Cytometrist. Make users confident about compensation, how to set voltage/gain, how to set gate, make them aware of basic ideas of flowcytometry, aware of what Flow Cytometry can do, what is new in the field.

Study material: flow-core resources & learning

Day 1: Basics of Flow Cytometry.

Day 2: Start with recap of day 1, answering questions if audience has any. Discussing what sorting is.

Introduction to compensation. Rules of compensation and Myths/Legends/Misconception of compensation.

Day 3: Recap and answering question if audience has any. Experiment

Day 4: Recap and answering question if audience has any. Experiment

Experiments we will offer:

  1. A four-color in-depth Apoptosis assay
  2. A four-color ROS assay using JC1, a Mitochondrial dye
  3. Intra cellular staining assay using different Cyclins and Ki67
  4. A classical PI based Cell cycle assay
  5. If user like to perform any other assay, we can discuss the feasibility

Contact: If you want to receive the training please send an email to bhowmickd20@ecu.edu

Day 1

  1. Few minutes on History of flow Cytometry up to 2021.
  2. What we can measure.
  3. Short comings of conventional flow.
  4. Different types of flow cytometer those are commercially available (Conventional system like BD, Acoustic flow like Attune, Imagestream, CyTOF and Spectral flow) explaining the pros and cons.
  5. Components of Flow Cytometer brief overview
    • Optics (why we use Laser, what is pinhole, explain DM, LP, BP and SP, how we use these filters to collect signal. Generation of scatter and fluorescence signal, obscuration bar.
    • Fluidics (Flow cell shape, Hydrodynamic focusing, laminar flow, effect of differential pressure on sample core size what does it mean for data quality), Laser delay
    • Electronics (explain generation of pulse, relation of H-A-W, explain how to use this information for singlet selection, shortly what ADC does and sampling frequency) and brief mentioning-type of detectors PMT, APD and CCD camera. Importance of Threshold. Linearity of a detector, why it is important using a cell cycle example.
  6. Different applications of Flow
  7. Very short introduction of fcs file.
  8. Use of different type of plots dot plot, contour plot and histogram, pros and cons.
  9. When to use linear and log display, what is bi exponential display.
  10. The reason we observe negative fluorescence.
  11. How to set gain (scatter & Fluorescence) with example
  12. Data analysis basic: Histogram vs dot plot, heat map or contour plot, why not histogram for cell cycle, part of population sort is actually whole, FMO needed, stimulated sample and FMO,
  13. Fluorochromes: How to match fluorochromes with BP filter and lasers, what stokes shift is, why it is important. Difference between primary dyes and tandem dyes. How degradation of fluorochromes can affect outcome.
  14. Effect of fixing on fluorochromes, spectra, separation BD data
  15. How conjugated Abs work, why we need blocking reagent, why good quality washing is important. Use of Isotype control, importance of viability dye, use time as quality control tool, centrifuge your Ab, Advantage of recombinant Ab
  16. Sample quality is the key, garbage in garbage out.

Day 2

  1. Recap of day 1 and answering any questions.
  2. Cleaning: why it is important?
  3. Sorting: Physical separation of particles, based on scatter or fluorescence, system charge the droplets, deflection plates moves those droplets to desired location. Cells can be sorted in bulk and in plates, 96, 384 or even 1536 well plate. Use of sorting.
  4. Introduction to unmixing/compensation
  5. Emission spectra
  6. Unwanted photons going to the detectors
  7. What is spill over
  8. Why spill over is a problem
  9. How to remove these unwanted signals
  10. Explain the rules of unmixing/compensation.
    • Use same fluorochrome in experiment and single stain
    • Single stains need to be bright or brighter than experiment sample
    • Positive and negative particles need to have same autofluorescence
    • Enough events to get a robust comp matrix
  11. What is detector linearity
  12. Why signals need to be within linearity range
  13. Single stains need to be treated same way as experimental sample
  14. Why washing is important for compensation
  15. Why every tandem dye needs to be considered as unique dye
  16. Myth:
    • Compensation introduces error.
    • One cannot compensate properly with beads, or one can only compensate properly with the same cells that are used in the experiment.
    • One cannot have compensation over 100%.
    • Same compensation setting can be used every day.
    • Compensation dependent on brightness.
  17. Why manual compensation is a bad approach
  18. Manual checking of the unmixing NXN plot
  19. High Dimensional data analysis
  20. Batch effect and barcoding

Day 3 & 4

  1. Recap compensation and answer any question.
  2. Basic hardware troubleshoots
  3. Experiments