Learn more about the pros and cons of 14 popular activation measurements, along with tips and recommendations for choosing the best method for your research. Access the full webinar recording with audio here: https://astartebio.com/resources/webinars/14-activation-methods/

1. Measuring Immune
Cell Activation
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2. • Immuno-oncology
• Vaccine development
• Autoimmune disease
• Infectious disease
Why Measure Immune Cell
Activation?

3. • T cells
o CD4+ T cells (helper)
o CD8+ T cells (cytolytic)
• B cells
• NK cells
• Myeloid cells
o Macrophages
o Dendritic cells
o Neutrophils
Immune Cells

5. • T and B cells will proliferate upon encounter with their
cognate antigen
• Requires engagement of the antigen receptor,
costimulatory receptor, and cytokines
• Many genes upregulated and cells develop distinct
phenotypes
Hallmarks of an Immune
Response

6. • Proliferation
• Cytokine Measurement
• Surface Antigen Expression
• Cytotoxicity
Categories of Assays

7. Proliferation Assays

8. • Traditional: 3H-thymidine incorporation
• Bromodeoxyuridine incorporation
• Tetrazolium dye (MTT, etc.)
• CFSE dye reduction
• CellTiter-Glo®
Proliferation Assay Choices

9. • Radiolabeled thymidine is
incorporated into DNA
during replication
• 3H is detected by liquid
scintillation
• Since most immune cells do
not make new DNA unless
they are activated, a
positive response is easily
detected against a low
background
3H-Thymidine
Uptake

10. Sample Data – 3H Thymidine

11. • 3H is radioactive
• Special instrument needed for detection of the signal
3H-Thymidine Drawbacks

12. • BrdU is an analog of
thymidine and is
incorporated into
replicating DNA
• Monoclonal antibody to
BrdU is used for detection
• Either ELISA format or
intracellular staining
Bromo-
deoxyuridine
Uptake

13. • Add BrdU to cell culture
• Incubate overnight (or longer)
• Remove culture medium and fix cells
• Permeabilize (depending on antibody/kit manufacturer)
• Add anti-BrdU
• Wash, add detection antibody labeled with peroxidase
• Wash, add substrate
ELISA Outline

14. Sample Data

15. Alternative Sample Data

16. • Add BrdU to cell culture
• Incubate overnight (or longer)
• Collect cells and stain surface antigens if desired
• Fix and permeabilize (depending on antibody/vendor)
• Add fluorescently labeled anti-BrdU
• Analyze by flow cytometry
Fluorescent Detection for Flow
Cytometry

17. Sample Data

18. • Fixation and permeabilization
• ELISA format takes time to develop
• Flow analysis can work better than fixation in a plate but
requires flow capability
Drawbacks to BrdU Methods

19. • Pros
o Both have great signal:noise ratio
o Familiar read-out (3H-thymidine)
• Cons
o Radioactivity
o Cumbersome because of need for fix-perm
S-Phase Methods

20. • MTT, XTT, et al.
• All rely on reduction of a substrate to a colored product by
mitochondrial enzymes
• Increased metabolic activity, increased cell numbers lead to
increased dye (color)
• None are sensitive enough in a primary readout
Tetrazolium Dye Methods

21. • Carboxyfluorescein diacetate succinimidyl ester (CFSE) and
its cousins
• Fluorescent label of cellular proteins
• With each cell division, individual cell fluorescence is
reduced by half
• Readout = flow cytometry
Dye Reduction Methods

24. Sample Data - CFSE

25. Co-Incubation of Responder Cells with Tregs but not
BC3 Cells Resulted in Suppression of Proliferation in
Response to Anti-CD3 Stimulation
CFSE
10
0
10
1
10
2
10
3
10
4
0
15
29
44
58
CFSE
10
0
10
1
10
2
10
3
10
4
0
12
25
37
49
CFSE
10
0
10
1
10
2
10
3
10
4
0
15
30
45
60
CFSE
10
0
10
1
10
2
10
3
10
4
0
15
30
45
60
CFSE
10
0
10
1
10
2
10
3
10
4
0
10
19
29
38
CFSE
10
0
10
1
10
2
10
3
10
4
0
20
40
59
79
CFSE
10
0
10
1
10
2
10
3
10
4
0
24
48
72
96
CFSE
10
0
10
1
10
2
10
3
10
4
0
31
63
94
125
CFSE
10
0
10
1
10
2
10
3
10
4
0
32
63
95
126
No Suppressor
1:16 1:8 1:4 1:2
Suppressor:Responder Ratio
Treg
BC3

26. • Pro
o Can combine with surface antigens to determine the
cell type that is proliferating
o Differences in the number of cell divisions can be
appreciated
• Con
o Takes more cells than other methods
o Not very high throughput
Pros and Cons

27. • Measures number of cells based on ATP content
• Proprietary firefly luciferase with 5 hour half-life
• Amount of luminescence directly related to the
number of cells present
• Sensitive detection of cells
CellTiter-Glo®

28. Sample Data – CellTiter-Glo®

29. More Sample Data
0
100000
200000
300000
400000
500000
600000
700000
800000
0 0.03 0.1 0.3 1
RLU
µg/mL tetanus peptide
Proliferative response of anti tetanus T cells

30. Cytokine Measurement

31. • Collection from culture medium
o Single cytokine or multiplex
o Timing of cytokine release
• Intracellular staining
o Identification of the cell population making cytokine
o Quantitation of reactive cells
Cytokine Measurement

32. Sample Data

33. Recall Assay Cytokines

34. • Antigen-presenting cells incubated with peptide overnight
• T cells added
• Co-culture continued overnight
• Added brefeldin A for 4 hours
• Stained dead cells and CD8
• Fixed and stained for IFNg
• 0.5% saponin used to permeabilize the cells
Intracellular Cytokine Staining

35. IFNg PE
Count
10
0
10
1
10
2
10
3
10
4
0
30
60
89
119
Intracellular Cytokine Staining
IFNg PE
Count
10
0
10
1
10
2
10
3
10
4
0
140
279
419
558
Antigen-Presenting CellsAntigen-Specific T Cells
Red histograms are co-cultures with irrelevant peptide
Black histograms are co-cultures with relevant peptide

36. • Pro
o Sensitive measure for both primary assays and
monitoring purified cells
• Con
o Requires initial workup to determine best timing
o Unless intracellular staining is done, the cells producing
cytokine can’t be identified
Pros and Cons of
Cytokine Assays

37. Activation Markers

38. • Some antigens are upregulated upon activation
o CD69
o CD25
o CD44
o Ki67
• Combining activation antigen can give detail into specific
cells participating in the activation (memory vs. naïve,
effector vs. memory)
Expression of Surface Antigens

39. Cytotoxicity

40. • Measurement of the ability of NK or T cells to kill target
cells (e.g., tumor cells, infected cells)
• Traditional methods label target cells with Cr51
• Nonradioactive methods suffer from high backgrounds and
inability to distinguish signal from target cells vs. cytotoxic
T cells
• Better methods needed
Cell-Mediated Cytotoxicity

41. • K562 transfected with
luciferase
• Cell killing can be measured
as release of luciferase
(increase in luciferase) or
loss of luciferase (longer
assay)
NK Lytic Assay
with K562-luc
0
2000
4000
6000
8000
10000
12000
14000
10 to 1 5 to 1 2.5 to 1 1.25 to 1 target alone
luminescence
Effector:target ratio
4 hours incubation – release of luc

42. • K562 targets labeled with
CFSE
• Incubate with NK cells
overnight
• Label with viability dye
(7-AAD)
• Analyze viability of CFSE
positive cells
NK Lytic Assay
Using Flow
Cytometric
Readout

43. • There are many ways to measure immunity
• Selection of a method depends on:
o Available instrumentation
o Number of samples to be analyzed
o Primary recall vs. polyclonal stimulation
o Need to identify the cells involved
Conclusions

44. Thank You