A New Robust Fluorescent Calcium Indicator for Ca2+ Flux Assays in Living Cells
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|
- 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).
ATP/Carbachol Induced Ca2+ Flux in CHO-K1 Cells with Probenecid
ATP/Carbachol Induced Ca2+ Flux in CHO-M1 Cells without Probenecid
RANTE Induced Ca2+ Flux in CHO-CCR5 Cells
Atropine Inhibited Ca2+ Flux in CHO-M1 Cells
Concanavalin A Induced CCE in Jurkat Cells
ATP Induced Ca2+ Flux by Flow Cytometry
- 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.
|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