Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays
Abstract
:1. Introduction
2. Experimental Section
2.1. Cells
2.2. ELISPOT Assay
2.3. Flow Cytometry Assay
2.4. ELISA
3. Results and Discussion
3.1. ELISPOT
Donor | Spots Per Well ± SD | Spot-Forming Cells (%) ± SD |
---|---|---|
1 | 326 ± 11.15 | 6.52 ± 0.23 |
2 | 155 ± 17.76 | 3.10 ± 0.36 |
3 | 47 ± 5.74 | 0.94 ± 0.12 |
4 | 23 ± 0.71 | 0.46 ± 0.02 |
5 | 143 ± 12.40 | 2.90 ± 0.23 |
3.2. ELISPOT vs. ELISA
Donor | ELISA (pg/mL) ± SD |
---|---|
1 | 212 ± 4.38 |
2 | 1449 ± 0.00 |
3 | 48 ± 2.46 |
4 | 268 ± 8.19 |
5 | 266 ± 1.803 |
3.3. ELISPOT vs. Flow Cytometry
Donor | Non-Stimulated Cells, % | Stimulated Cells, % | ||||
---|---|---|---|---|---|---|
CD3 Negative Cells | T-cells | All Cells | CD3 Negegative Cells | T-Cells | All Cells | |
1 | 0.61 | 0.65 | 1.26 | 21.0 | 12.40 | 33.40 |
2 | 0.33 | 0.50 | 0.83 | 18.50 | 13.80 | 32.30 |
3 | 1.18 | 0.75 | 1.93 | 16.50 | 6.54 | 23.04 |
4 | 1.02 | 0.77 | 1.79 | 8.29 | 7.83 | 16.12 |
5 | 0.59 | 0.74 | 1.33 | 7.84 | 15.50 | 23.34 |
Cells | Rankings of Donors |
---|---|
Non-stimulated CD3 neg. cells | 3 > 4 > 1 > 5 > 2 |
Non-stimulated T-cells | 4 > 3 > 5 > 1 > 2 |
All non-stimulated cells | 3 > 4 > 5 > 1 > 2 |
Stimulated CD3 neg. cells | 1 > 2 > 3 > 4 > 5 |
Stimulated T-cells | 5 > 2 > 1 > 4 > 3 |
All stimulated cells | 1 > 2 > 5 > 3 > 4 |
Donor | Stimulated Cells | Fraction of IFNγ- Secreting Cells (%) | |
---|---|---|---|
Flow Cytometry (“All Cells”, %) | ELISPOT (SFCs ± SD, %) | ||
1 | 33.4 | 6.52 ± 0.23 | 19.52 |
2 | 32.3 | 3.1 ± 0.36 | 9.6 |
3 | 23.04 | 0.94 ± 0.12 | 4.08 |
4 | 16.12 | 0.46 ± 0.02 | 2.85 |
5 | 23.34 | 2.9 ± 0.23 | 4.28 |
3.4. ELISA vs. Flow Cytometry
3.5. Discussion
Donor | Normalized Values | ||
---|---|---|---|
ELISPOT | Flow Cytometry(All stimulated cells) | ELISA | |
1 | 14.17 | 2.07 | 4.42 |
2 | 6.74 | 2.00 | 30.19 |
3 | 2.04 | 1.43 | 1.00 |
4 | 1.0 | 1.00 | 5.58 |
5 | 6.30 | 1.45 | 5.54 |
Rankings of Donors | 1 > 2 > 5 > 3 > 4 | 1 > 2 > 5 > 3 > 4 | 2 > 4 ≥ 5 > 1 > 3 |
4. Conclusions
Acknowledgments
Author contributions
Conflicts of Interest
References
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Hagen, J.; Zimmerman, R.; Goetz, C.; Bonnevier, J.; Houchins, J.P.; Reagan, K.; Kalyuzhny, A.E. Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays. Cells 2015, 4, 84-95. https://doi.org/10.3390/cells4010084
Hagen J, Zimmerman R, Goetz C, Bonnevier J, Houchins JP, Reagan K, Kalyuzhny AE. Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays. Cells. 2015; 4(1):84-95. https://doi.org/10.3390/cells4010084
Chicago/Turabian StyleHagen, Jodi, Ryan Zimmerman, Christine Goetz, Jody Bonnevier, Jeffrey P. Houchins, Kevin Reagan, and Alexander E. Kalyuzhny. 2015. "Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays" Cells 4, no. 1: 84-95. https://doi.org/10.3390/cells4010084