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

Principle of Calbryte™ 520 Am fluorescent calcium indicator.

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.

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

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.
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.
Cells were then incubated with or without compounds of interest for additional 15 min at 37°C.
The dye loading buffer was either removed and replace with 200 µL HHBS (wash condition) or was left in wells (no-wash condition).
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

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

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

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

Calbryte™ 520 IC50 (nM): 0.48
Fluo-4 IC50 (nM): <100

Calbryte™ 520 IC50 (nM): 1.54
Fluo-4 IC50 (nM): 2.01

Calbryte™ 520 IC50 (nM): 1.1
Fluo-4 IC50 (nM): 2.12

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

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

B

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

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.
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.
The calculated EC₅₀ of Ca²⁺ flux stimulants or inhibitors were comparable between Calbryte™ 520 AM and Fluo-4 AM incubated cells.

Ordering Information

Table 2. Calbryte™ 520 green fluorescent calcium indicators for live cell calcium imaging.

Indicator ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Filter Set ▲ ▼	Kd¹ ▲ ▼	Φ² ▲ ▼	FCa/FFree³ ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
Calbryte™ 520 AM	493	515	FITC	1.2 µM	0.75	∼300 fold	2x50 µg	20650
Calbryte™ 520 AM	493	515	FITC	1.2 µM	0.75	∼300 fold	10x50 µg	20651
Calbryte™ 520 AM	493	515	FITC	1.2 µM	0.75	∼300 fold	1 mg	20653
Calbryte™ 520, potassium salt	493	515	FITC	1.2 µM	0.75	∼300 fold	10x50 µg	20658
Calbryte™ 520, potassium salt	493	515	FITC	1.2 µM	0.75	∼300 fold	2x50 µg	20656

Table 3. Fluo-4 green fluorescent calcium indicators for live cell calcium imaging.

Indicator ▲ ▼	Ex (nm) ▲ ▼	Em (nm) ▲ ▼	Filter Set ▲ ▼	Kd¹ ▲ ▼	Φ² ▲ ▼	FCa/FFree³ ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	FITC	345 nM	0.16	∼100 fold	1 mg	20550
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	FITC	345 nM	0.16	∼100 fold	10x50 µg	20551
Fluo-4 AM Ultrapure Grade CAS 273221-67-3	495	528	FITC	345 nM	0.16	∼100 fold	5x50 µg	20552
Fluo-4, Pentapotassium Salt	495	528	FITC	345 nM	0.16	∼100 fold	1 mg	20555
Fluo-4, Pentapotassium Salt	495	528	FITC	345 nM	0.16	∼100 fold	1 mg	20556

Table 4. Probenecid and Pluronic® F-127 products for improving calcium indicator retention and aqueous solubility.

Product ▲ ▼	Unit Size ▲ ▼	Cat No. ▲ ▼
ReadiUse™ probenecid 25 mM stabilized aqueous solution	10x10 mL	20062
ReadiUse™ probenecid, sodium salt Water-soluble	10x77 mg	20061
Probenecid Cell culture tested CAS 57-66-9	10x72 mg	20060
Pluronic® F-127 20% solution in DMSO	10 mL	20052
Pluronic® F-127 Cell culture tested	10 g	20050
Pluronic® F-127 10% solution in water	10 mL	20053