This document provides an overview of flow cytometry techniques and applications. It discusses experimental controls used in flow cytometry including unstained, isotype, internal negative, positive, compensation and viability controls. It describes how flow cytometry can be used to analyze surface and intracellular markers, immune responses, cell proliferation, migration, suppression potential and more. Specific examples are given on analyzing peripheral blood mononuclear cells, lung tissue, lymph nodes and studying CD4+CD25+ T cell suppression and migration. Guidance is provided on reporting flow cytometry data, controlling for autofluorescence, validating results against literature, and properly analyzing the large amount of data generated.

1. Part 1: Technical protocols
for Flow Cytometry and its
application
Rohimah Mohamud, PhD
Immunology Department
Medical School, University Sains Malaysia
rohimahm@usm.my

2. Richard R. Jahan-Tigh et al., Journal of Investigative Dermatology, 2012, 132

3. Richard R. Jahan-Tigh et al., Journal of Investigative Dermatology, 2012, 132

4. Experimental (Staining) controls
• Unstained control
• Isotype control
• Internal negative control
• Positive control
• Compensation controls
• Viability control
• Fc blocking control

5. What can we do with flow cytometry….
• Surface markers and intracellular markers
• innate and adaptive immune responses
• cellular and humoral immune responses (cytokine)
• Suppression potential
• Migration potential
• Functional capacity of cells
• Proliferation potential (Ki-67 marker)
• Survival potential
• Cell cycling with CFSE staining
• Neutrophil oxidative burst (Measurement of ROS)
• Kinase phosphorylation
• Degranulation
• correlate with in vitro studies as well as microscopic
images

6. Surface markers for Peripheral vs lymphoid organs
FSC
SSC
PBMC BAL Lung LN
CD4
CD3
Source: Rohimah’s unpublished data

7. Suppression and Migration potential of CD4+CD25+ T cells
We can correlate with in vitro studies:
• Suppression assay
• Migration assay
Source: Rohimah’s unpublished data

9. Humoral immune responses (cytokine)
Pieper et al., BMC Immunology 14(1):34, 2013
Stain cells
Stimulate and treat
with protein and
transport inhibitor
Fix and
permeabilize cells
Flow
Cytometry
analysis

10. Test
group
Negative
control
Gated from
live cells
Gated from
lymphocytes
CFSE staining for proliferation assay
Only lymphocytes responded to the antigen and proliferated!!
Source: Rohimah’s unpublished data

11. Ki-67… a proliferating marker
extensively used as a prognostic and
predictive marker for cancer diagnosis
Lung-draining LN
Subset of cells #1
Subset of cells #2
Subset of cells #3
Source: Rohimah’s unpublished data

12. https://www.caymanchem.com/pdfs/601130.pdf

13. Need to know the characteristics
of your cells….
• Autofluorescence cells (especially from lungs)
• Always compare your cell populations with reliable
journals
• Ask the experts about compensation set up and
gating strategy

14. How to report your data…
• Percentages of cells
• Number of cells
• Median Fluorescence Intensity (MFI)
• Ratio
• Correlation with other cells/cytokines
Massive amount of data….this is good!!
BUT you need to know how to analyze your data

15. Thank you for your attention
Any questions please email rohimahm@usm.my