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A New Robust Fluorescent Calcium Indicator for Ca2+ Flux Assays in Living Cells

by Haitao Guo, Jinfang Liao, Qin Zhao, Ruogu Peng, Zhenjun Diwu, Grace Yang

Introduction


Fluorescent Ca2+ indicators (e.g. Indo-1, Fura-2, Fluo-3 and Fluo-4) are commonly used to monitor Ca2+ response in a variety of applications including HTS, fluorescent imaging and flow cytometry. However, these dyes require the presence of an organic anion transporter inhibitor (e.g. probenecid) to prevent the leakage of the indicators in many cell types (e.g. CHO and Hela). The organic anion transporter inhibitors are toxic to cells and some of them are known to be the inhibitors of certain GPCRs (e.g. chemokine receptors, bitter taste receptors). Because of those reasons, the usages of these dyes are limited.

We have recently developed the first-in-class ultra bright probenecid-free fluorescent Ca2+ indicator, Calbryte™ 520 AM. Calbryte™ 520 AM is cell permeable and non-fluorescent. Once inside the cells, Calbryte™ 520 AM is processed into Calbryte™ 520 by esterases. When binding to Ca2+, Calbryte™ 520 produces exceptionally bright fluorescent signal with very high signal to background (S/B) ratio.

 

Calbryte™mechanism
Figure 1. Principle of Calbryte™ 520 Am fluorescent calcium indicator.

 

Table 1. Calbryte™ 520 AM specifications.

Indicator Permeability Ex (nm) Em (nm) Kd Unit Size Cat No.
Calbryte™ 520 AM Cell Permeant 490 nm 525 nm 1.2 µM 10x50 µg 20651



Method


  1. 100 µL/well cells (40K/well for adherent cells) were seeded in a 96-well black wall/clear bottom Costar plate at 37°C incubator overnight.
  2. The cells were incubated with 100 µL dye loading buffers that contain Ca2+ indicators (5 µg/mL) at 37°C for 30 minutes to 1 hour.
  3. Cells were then incubated with or without compounds of interest for additional 15 min at 37°C.
  4. The dye loading buffer was either removed and replace with 200 µL HHBS (wash condition) or was left in wells (no-wash condition).
  5. 50 µL/well of Ca2+ flux stimulant was added and the Ca2+ flux was monitored by Fluorescence Microscope (Keyence), FlexStation (Molecular Devices) or Flow Cytometer (Novocyte 3000).



Results


ATP/Carbachol Induced Ca2+ Flux in CHO-K1 Cells with Probenecid

Calcium Response in CHO-K1
Figure 2. ATP induced endogenous P2Y response in CHO-K1 cells. Calbryte™520 AM or Fluo-4 AM in Pluronic F127 containing buffer with 2.5 mM probenecid was incubated with the cells at 37°C for 45 min. 10 µM ATP (final conc.) was added, and images were taken by a fluorescence microscope using FITC channel.

 

ATP/Carbachol Induced Ca2+ Flux in CHO-M1 Cells without Probenecid

Calcium Response in CHO-M1
Figure 3. ATP induced endogenous P2Y response in CHO-K1 cells. Calbryte™520 AM or Fluo-4 AM in Pluronic F127 containing buffer with 2.5 mM probenecid was incubated with the cells at 37°C for 45min. 10 µM ATP (final conc.) was added, and images were taken by a fluorescence microscope using FITC channel.

 

RANTE Induced Ca2+ Flux in CHO-CCR5 Cells

Figure 3A
Figure 3B
Figure 3C
Figure 4. CHO cells that express chemokine receptor 5 (CCR5) were incubated with Calbryte™520 AM or Fluo-4 AM for 1 hour in different dye loading buffers at 37 °C. A) HHBS with Pluronic F127 without probenecid. B) HHBS with Pluronic F127 with 2.5 mM probenecid. C) HHBS with Pluronic F127 Plus buffer without probenecid. Intracellular Ca2+ was monitored before and after the addition of 100 nM RANTES (final conc.) via FlexStation for 100s.

 

Atropine Inhibited Ca2+ Flux in CHO-M1 Cells

Figure 4A
  Calbryte™ 520 Fluo-4
IC50 (nM) 0.48 >100
Figure 4B
  Calbryte™ 520 Fluo-4
IC50 (nM) 1.54 2.01
Figure 4C
  Calbryte™ 520 Fluo-4
IC50 (nM) 1.1 2.12
Figure 5. CHO cells that express M1 muscarinic receptor were incubated with Calbryte™520 AM or Fluo-4 AM in different loading buffers for 1 hour at 37°C. A) HHBS with Pluronic F127 without probenecid. B) HHBS with Pluronic F127 with 2.5 mM probenecid. C) HHBS with Pluronic F127 Plus buffer without probenecid. Atropine was added to the cells and incubated for additional 15 min at 37°C. Intracellular Ca2+ was monitored before and after the addition of 1 µM carbachol ( final conc.) via FlexStation for 100s.

 

Concanavalin A Induced CCE in Jurkat Cells

Figure5
Figure 6. 250K Jurkat cells were suspended in calcium-free HHBS buffer per well, cells were incubated with Calbryte™520 AM or Fluo-4 AM in HHBS with Pluronic F127 Plus for 1 hr at 37°C. 3 µg/ml of channel opener concanavalin A (final conc.) with or without channel inhibitor capsaicin were incubated with the cells for 10 min at 37°C. The capacitive Ca2+ entry (CCE) was monitored before and after the addition of 5 mM CaCl2 via FlexStation for 100s.

 

ATP Induced Ca2+ Flux by Flow Cytometry

A
Figure 6A
Figure 6B
Figure 6C
Figure 6D

B

Figure 7A
Figure 7B
Figure 7. CHO-K1 cells were incubated with Calbryte™ 520 AM or Fluo-4 AM in HHBS with Pluronic F127 and 2.5 mM probenecid for 30 min at 37°C. The cells were detached from the wells and washed twice with HHBS. The baseline was acquired prior to the addition of ATP and after the addition of ATP over time by Flow Cytometer. A) 0 µM, 1 µM or 10 µM ATP were added to the cells. The arrows on the graph indicate the time between addition of ATP and the actual analysis. B) Time-dependent changes of fluorescent signal. Time 0 is the ATP stimulation time, and the initial detection was ~30s relative to stimulation.



Conclusion


  1. Calbryte™520 AM generated much brighter signal (3-4 fold more) and much higher S/B ratio (3-4 folds more) in monitoring Ca2+ flux in CHO-K1, CHO-M1, CHO-CCR5 and Jurkat cells than Fluo-4 AM using fluorescent microscope, FlexStation and Flow Cytometer.
  2. Without probenecid, Fluo-4 AM was unable to detect Ca2+ flux in CHO cells, however, Calbryte™520 AM still achieved excellent cellular retention and S/B ratio.
  3. The calculated EC50 of Ca2+ flux stimulants or inhibitors were comparable between Calbryte™520 AM and Fluo-4 AM incubated cells.



Ordering Information

Product Unit Size Cat No.
Calbryte™ 520 AM 2x50 µg 20650
Calbryte™ 520 AM 10x50 µg 20651
Calbryte™ 520 AM 1 mg 20653
Fluo-4 AM 5x50 µg 20552
Fluo-4 AM 10x50 µg 20551
Fluo-4 AM 1 mg 20550
HHBS [Hanks' Buffer with 20 mM Hepes] 100 mL 20011
Pluronic® F-127 *Cell Culture Tested* 10 g 20050
Pluronic® F-127 *20% solution in DMSO* 10 mL 20052
Pluronic® F-127 *10% solution in water* 10 mL 20053
Probenecid *Cell culture tested, CAS 57-66-9* 10x72 mg 20060
ReadiUse™ Probenecid *25 mM stabilized aqueous solution* 10x10 mL 20062
ReadiUse™ Probenecid, sodium salt *Water-soluble* 10x77 mg 20061


Original created on November 21, 2020, last updated on November 21, 2020
Tagged under: Calcium Imaging, Calcium GPCR Analysis, Calcium Imaging, Calcium Mobilization