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Calcein Deep Red™ AM ester
Calcein AM is one the most popular fluorescent probes used for labeling and monitoring cellular functions of live cells. However, the single color of Calcein AM makes it impossible to use this valuable reagent in the multicolor applications. For example, it is impossible to use Calcein AM in combination of GFP-tranfacted cells due to the same color to GFP. To address this color limitation of Calcein AM, we have developed Calcein Orange™, Calcein Red™ and Calcein Deep Red™. These new Calcein AM ester enables the multicolor labeling and functional analysis of live cells in combination with Calcein AM. Non-fluorescent Calcein Deep Red™ AM ester can easily get into live cells and hydrolyzes to generate strongly fluorescent Calcein Deep Red™ (Cat#: 21902) dye. Calcein Deep Red™ dye can be monitored with the common Cy5 filter set.
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
Calcein Deep Red™ AM ester stock solution
Prepare a 2 to 5 mM stock solution of Calcein Deep Red™ AM in high-quality, anhydrous DMSO.Note The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
PREPARATION OF WORKING SOLUTION
Calcein Deep Red™ AM ester working solution
Prepare a Calcein Deep Red™ AM working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein Deep Red™ AM at the final concentration of 4 to 5 µM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.Note If your cells contain organic anion-transporters, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the working solution to reduce leakage of the de-esterified indicators.
SAMPLE EXPERIMENTAL PROTOCOL
- Prepare cells for imaging.
- Remove the cell culture medium and wash cells once with serum-free buffer to remove any remaining media.
Note Serum in cell culture media may contain esterase activity, which can increase background interference. - Add Calcein Deep Red™ AM working solution to the culture.
- Incubate cells at 37 °C for 30 to 60 minutes.
- Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
- Measure the fluorescence intensity using either a fluorescence microscope equipped with a Cy5 filter set, a flow cytometer equipped with a 660/20 nm filter (APC channel), or a fluorescence plate reader at Ex/Em = 620/660 nm cutoff 630 nm.
Spectrum
Citations
View all 35 citations: Citation Explorer
Directing CAR NK Cells via the Metabolic Incorporation of CAR Ligands into Malignant Cell Glycans
Authors: Antillon, Kathia and Ross, Patrick A and Farrell, Mark P
Journal: ACS Chemical Biology (2022)
Authors: Antillon, Kathia and Ross, Patrick A and Farrell, Mark P
Journal: ACS Chemical Biology (2022)
Radioiodination of extravesicular surface constituents to study the biocorona, cell trafficking and storage stability of extracellular vesicles
Authors: Yerneni, Saigopalakrishna S and Solomon, Talia and Smith, Jason and Campbell, Phil G
Journal: Biochimica et Biophysica Acta (BBA)-General Subjects (2022): 130069
Authors: Yerneni, Saigopalakrishna S and Solomon, Talia and Smith, Jason and Campbell, Phil G
Journal: Biochimica et Biophysica Acta (BBA)-General Subjects (2022): 130069
A ratiometric fluorescent assay for the detection and bioimaging of alkaline phosphatase based on near infrared Ag2S quantum dots and calcein
Authors: Cai, M., Ding, C., Wang, F., Ye, M., Zhang, C., Xian, Y.
Journal: Biosens Bioelectron (2019): 148-153
Authors: Cai, M., Ding, C., Wang, F., Ye, M., Zhang, C., Xian, Y.
Journal: Biosens Bioelectron (2019): 148-153
Ratio fluorometric determination of ATP base on the reversion of fluorescence of calcein quenched by Eu(III) ion using carbon dots as reference
Authors: Zhang, C., Zhang, H., Yu, Y., Wu, S., Chen, F.
Journal: Talanta (2019): 451-456
Authors: Zhang, C., Zhang, H., Yu, Y., Wu, S., Chen, F.
Journal: Talanta (2019): 451-456
Factors Affecting the Acoustic In Vitro Release of Calcein from PEGylated Liposomes
Authors: Ahmed, S. E., Moussa, H. G., Martins, A. M., Abbas, Y., Al-Sayah, M. H., Husseini, G. A.
Journal: J Nanosci Nanotechnol (2019): 6899-6906
Authors: Ahmed, S. E., Moussa, H. G., Martins, A. M., Abbas, Y., Al-Sayah, M. H., Husseini, G. A.
Journal: J Nanosci Nanotechnol (2019): 6899-6906
Page updated on December 3, 2024
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