NovoCyte Flow Cytometer - ACEA Biosciences · For life science research only. Not for use in...
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Application Note No. 17 © 2017 ACEA Biosciences, Inc. All rights reserved. 6779 Mesa Ridge Road Ste 100 San Diego, CA 92121 | 866.308.2232 | www.aceabio.com App Note No. 17 NovoCyte ® Flow Cytometer Calcium (Ca 2+ ) is an important secondary messenger and a key component of many cell processes. It is an extremely versale molecule used by all cells for various signaling pathways, occurring during the acvaon of G-Protein coupled receptors, synapc transmission of neurons, muscle contracon, and angen specific acvaon of T cells. Unlike many other secondary messengers, Ca 2+ is not synthesized or metabolized, but stored and released by channels/pumps that maintain Ca 2+ concentraons in disnct cellular compartments. Alteraons in intracellular Ca 2+ concentraons are amongst the most rapid cellular responses, ranging from faster than a microsecond to several minutes depending on the specific smuli. Methods that are currently used to detect Ca 2+ flux include confocal microscopy, plate based assays, and flow cytometry. Flow cytometry has the advantage of the unique capability to determine Ca 2+ levels in several millions of individual cells over me as well as in an enre populaon. Intracellular Ca 2+ measurements ulize fluorescent Ca 2+ indicators which exhibit an increase in fluorescence upon binding to Ca 2+ . Ca 2+ indicators such as Quin-2, Fura-2, Indo-1, Fluo-3, Fluo-4/AM can be used to quanfying Ca 2+ concentraons by flow cytometry. In this applicaon note, calcium flux was measured in a cultured T cell line using the NovoCyte flow cytometer to assess the inhibion of T cell receptor (TCR) -mediated calcium flux by Ibrunib. Inhibion of T Cell Receptor Signaling Mediated Calcium Flux in T cells Calcium flux in Jurkat T cells on the NovoCyte flow cytometer Increased intracellular Ca 2+ content is an essenal and irreversible step towards the acvaon of T lymphocytes and indicated in regulang their cell survival, proliferaon and differenaon. Upon angen recognion, acvaon of the TCR iniates numerous signaling cascades resulng in cellular acvaon and proliferaon. During TCR signaling, IL-2-inducible kinase (ITK) acvates phospholipase C (PLC) and the subsequent release of intracellular Ca 2+ stores from the endoplasmic reculum (ER) resulng in a rapid elevaon of intracellular Ca 2+ levels up to 500nM. This process subsequently triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca 2+ , which in turn acvates key molecules such as Calcineurin. Inhibion of calcium flux has been shown to affect T cell funcon, therefore, measuring the Ca 2+ content in T cells presents important informaon about the status and kinecs of T cell acvaon. The capability of the NovoCyte flow cytometer to quanfy dynamic changes in intracellular Ca 2+ was demonstrated by measuring calcium flux in Jurkat T cells. Calcium flux was induced by A23187 or Ionomycin, both effecve Ca 2+ carriers that increase the ability of divalent ions to cross biological membranes. Resng Jurkat T cells were loaded with the Ca 2+ indicator dye, Fluo-4/AM. Fluo-4/AM binds to free Ca 2+ , and the intensity of the fluorescence signal is proporonal to the concentraon. Ca 2+ concentraon was measured by the change in Fluo- 4/AM fluorescence over me; a stable background of Fluo-4/AM is seen before the addion of calcium flux smulants (Figure 1A-C, leſt secon of plot). Upon addion of A23187 or Ionomycin, mean fluorescence signal increases immediately, indicang an elevaon in intracellular Ca 2+ concentraon. Notably, the effect of A23187 is more robust than the addion of Ionomycin (Figure 1B-D). This data demonstrates that dynamic alteraons in intracellular Ca 2+ concentraon can be measured on the NovoCyte flow cytometer. Figure 1. Calcium flux in Jurket cells. Jurkat T cells were loaded with Fluo-4/AM and acquired on the NovoCyte flow cytometer. Baseline for each sample was acquired and the rest of the cells were analyzed aſter the addion of Ca+ influx smulants and response measured over me. A. DMSO B. 2.5μM A23187; C. 2μg/ mL of Ionomycin ; D. Time-dependent changes of intracellular Ca 2+ concentraon. Data was normalized to baseline by the equaon of Relave Intracellular Ca2+ (RIC) = fold of baseline (inial stage) - 1. Time points depicted on X axis are the me relave to smulaon. Zero is the smulaon me, and the inial detecon point was ~ 20s relave to simulaon. Data was normalized to baseline (n = 3, means +/- SEM). Time relave to smulaon (minutes) Relave Intracellular Ca 2+ A B C DMSO A23187 Ionomycin D DMSO 2.5 μM A23187 2μg/mL Ionomycin
Transcript of NovoCyte Flow Cytometer - ACEA Biosciences · For life science research only. Not for use in...
Application Note No. 17
© 2017 ACEA Biosciences, Inc. All rights reserved. 6779 Mesa Ridge Road Ste 100 San Diego, CA 92121 | 866.308.2232 | www.aceabio.com
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NovoCyte® Flow Cytometer
Calcium (Ca2+) is an important secondary messenger and a key component of many cell processes. It is an extremely versatile molecule used by all cells for various signaling pathways, occurring during the activation of G-Protein coupled receptors, synaptic transmission of neurons, muscle contraction, and antigen specific activation of T cells. Unlike many other secondary messengers, Ca2+ is not synthesized or metabolized, but stored and released by channels/pumps that maintain Ca2+ concentrations in distinct cellular compartments. Alterations in intracellular Ca2+ concentrations are amongst the most rapid cellular responses, ranging from faster than a microsecond to several minutes depending on the specific stimuli. Methods that are currently used to detect Ca2+ flux include confocal microscopy, plate based assays, and flow cytometry. Flow cytometry has the advantage of the unique capability to determine Ca2+ levels in several millions of individual cells over time as well as in an entire population. Intracellular Ca2+ measurements utilize fluorescent Ca2+ indicators which exhibit an increase in fluorescence upon binding to Ca2+. Ca2+ indicators such as Quin-2, Fura-2, Indo-1, Fluo-3, Fluo-4/AM can be used to quantifying Ca2+ concentrations by flow cytometry. In this application note, calcium flux was measured in a cultured T cell line using the NovoCyte flow cytometer to assess the inhibition of T cell receptor (TCR) -mediated calcium flux by Ibrutinib.
Inhibition of T Cell Receptor Signaling Mediated Calcium Flux in T cells
Calcium flux in Jurkat T cells on the NovoCyte flow cytometer
Increased intracellular Ca2+ content is an essential and irreversible step towards the activation of T lymphocytes and indicated in regulating their cell survival, proliferation and differentiation. Upon antigen recognition, activation of the TCR initiates numerous signaling cascades resulting in cellular activation and proliferation. During TCR signaling, IL-2-inducible kinase (ITK) activates phospholipase C (PLC) and the subsequent release of intracellular Ca2+ stores from the endoplasmic reticulum (ER) resulting in a rapid elevation of intracellular Ca2+ levels up to 500nM. This process subsequently triggers the opening of ion channels in the plasma membrane allowing an influx of extracellular Ca2+, which in turn activates key molecules such as Calcineurin. Inhibition of calcium flux has been shown to affect T cell function, therefore, measuring the Ca2+ content in T cells presents important information about the status and kinetics of T cell activation. The capability of the NovoCyte flow cytometer to quantify dynamic changes in intracellular Ca2+ was demonstrated by measuring calcium flux in Jurkat T cells. Calcium flux was induced by A23187 or Ionomycin, both effective Ca2+ carriers that increase the ability of divalent ions to cross biological membranes. Resting Jurkat T cells were loaded with the Ca2+ indicator dye, Fluo-4/AM. Fluo-4/AM binds to free Ca2+, and the intensity of the fluorescence signal is proportional to the concentration. Ca2+ concentration was measured by the change in Fluo-4/AM fluorescence over time; a stable background of Fluo-4/AM is seen before the addition of calcium flux stimulants (Figure 1A-C, left section of plot). Upon addition of A23187 or Ionomycin, mean fluorescence signal increases immediately, indicating an elevation in intracellular Ca2+ concentration. Notably, the effect of A23187 is more robust than the addition of Ionomycin (Figure 1B-D). This data demonstrates that dynamic alterations in intracellular Ca2+ concentration can be measured on the NovoCyte flow cytometer.
Figure 1. Calcium flux in Jurket cells. Jurkat T cells were loaded with Fluo-4/AM and acquired on the NovoCyte flow cytometer. Baseline for each sample was acquired and the rest of the cells were analyzed after the addition of Ca+ influx stimulants and response measured over time. A. DMSO B. 2.5μM A23187; C. 2μg/mL of Ionomycin ; D. Time-dependent changes of intracellular Ca2+ concentration. Data was normalized to baseline by the equation of Relative Intracellular Ca2+ (RIC) = fold of baseline (initial stage) - 1. Time points depicted on X axis are the time relative to stimulation. Zero is the stimulation time, and the initial detection point was ~ 20s relative to simulation. Data was normalized to baseline (n = 3, means +/- SEM).
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For life science research only. Not for use in diagnostic procedures.
© 2017 ACEA Biosciences, Inc. All rights reserved. 6779 Mesa Ridge Road Ste 100 San Diego, CA 92121 | 866.308.2232 | www.aceabio.comNovoCyte and NovoExpress are trademarks of ACEA Biosciences, Inc. in the US and other countries. All other product names and trademarks are the property of their respective owners.
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Inhibition of CD3-mediated calcium flux in Jurkat T cells by Ibrutinib
1. Berridge, M.J., Lipp, P., Bootman, M.D., 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1 (1), 11.
2. Feske, S., 2007. Calcium signaling in lymphocyte activation and disease. Nat. Rev. Immunol. 7, 690–702.3. Smith-Garvin, J.E., Koretzky, G.A., Jordan, M.S., 2009. T cell activation. Annu. Rev. Immunol. 27, 591.4. Parekh, A.B., 2007. Functional consequences of activating store-operated CRAC channels. Cell Calcium 42, 111–121.5. Lewis RS, Cahalan MD., 1989. Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human
leukemic T cells. Cell Regul 1:99–112.6. C.S. Guy, D.A. Vignali Organization of proximal signal initiation at the TCR:CD3 complex Immunol. Rev., 232 (2009), pp. 7–217. Dubovsky, JA., Kyle A. Beckwith, et al, 2013. Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective
pressure in T lymphocytes. Blood,2013;122(15):2539-2549
References
Conclusion
Accurate measurements of intracellular Ca2+ concentration allows for a more comprehensive understanding of Ca2+ regulated cell functions and signaling pathways that affect a wide range of physiological and pathological processes. Calcium flux can be induced by Ca2+ stimulants and measured on the NovoCyte flow cytometer to allow real time Ca2+ measurement at the single cell level. The NovoCyte flow cytometer offers high performance flow cytometry making the application of calcium flux assays and related studies easy to perform.
T cell calcium flux is initiated by TCR signaling leading to a sequence of events culminating in the release of Ca2+ stores from the ER and influx of extracellular Ca2+ into the cell. Ibrutinib is a small molecule that specifically blocks TCR signaling by inhibition of IL-2-inducible kinase (ITK). We used the NovoCyte flow cytometer to investigate the inhibitory effect of Ibrutinib on TCR activation through calcium flux induced by CD3 crosslinking. Addition of αCD3 antibody results in a rapid increase of Ca2+ in Jurkat T cells attaining an intracellular Ca2+
concentration 3.5 fold higher than the baseline (Figure 2). Peak Ca2+ concentrations are reached about 1.5 minutes after addition of αCD3 antibody (Figure 2D). Next, the effect of the Ibrutinib on calcium flux was assayed; Jurkat T cells were either pretreated with DMSO or Ibrutinib. Treament with DMSO alone had no effect on TCR-induced calcium flux in Jurket T cells (Figure 2B). However, calcium flux was completely abolished with Ibrutinib (Figure 2C). By measuring the kinetics of Ca2+ mobilization on the NovoCyte flow cytometer we can monitor defects or inhibition of TCR signaling, as was shown with Ibrutinib treatment.
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Figure 2. Ibrutinib inhibition of CD3-mediated calcium flux in Jurkat T cells. Jurkat T cells were loaded with Fluo-4/AM and then treated with DMSO or 1 μM Ibrutinib. Baseline was acquired than the rest of the cells were stimulated with 5 μg/mL anti-CD3 and further analyzed. A. Without treatment; B. Treated with DMSO; C. Treated with 1μM Ibrutinib; D. Time-dependent changes of intracellular Ca2+ concentration. Data was normalized to baseline by the equation of Relative Intracellular Calcium (RIC) = fold of baseline (initial stage) - 1. Time points depicted on X axis are the time relative to stimulation. Zero is the stimulation time, and the initial detection point was ~20s relative to simulation. Data was normalized to baseline (n = 3, means +/- SEM).
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