ReadiUse™ Preactivated APC-Cy7 Maleimide

Flow cytometry analysis of whole blood stained with APC-Cy7 anti-human CD8 *HIT8a* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the APC-Cy7 specific R7-A channel.

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Molecular weight	N/A
Solvent	Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	779

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Refrigerated (2-8 °C); Minimize light exposure

Overview SDS Protocol

Molecular weight

N/A

Extinction coefficient (cm ^-1 M ^-1)

700000

Excitation (nm)

651

Emission (nm)

779

APC-Cy7 is a popular tandem color used in flow cytometry. Its primary absorption peak is at 651 nm with emission peak at ~780 nm. AAT Bioquest offers this preactivated APC-Cy7 to facilitate the APC-Cy7 tandem conjugations to reduced antibodies and other biomolecules that contain a thiol group. Our preactivated APC-Cy7 maleimide is prepared from the commonly used crosslinker SMCC, and ready to conjugate. Allophycocyanin (APC) is a phycobiliprotein isolated from Spirulina sp., a blue-green alga. Like other phycobiliproteins, APC is fluorescent, with an extremely high absorptivity and a high quantum efficiency. It is a protein which can be easily linked to antibodies and other proteins by conventional protein cross-linking techniques without altering its spectral characteristics.

Example protocol

SAMPLE EXPERIMENTAL PROTOCOL

Reduction of Antibody

Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody solutions should be at 2 mg/mL or higher for best results. The reduction can be carried out in different buffers for example: MES, phosphate, and TRIS buffers (pH range 6 to 8). The antibody should be concentrated if less than 2 mg/mL.
Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution and mix well. Let the antibody solution stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Purify the reduced antibody over a desalting column pre-equilibrated with 50 mM MES Buffer (pH=6.0-6.5) with 2 mM EDTA. (Desalting column: https://www.aatbio.com/products/readiuse-bio-gel-p-6-spin-column?unit=60500)
Measure the Antibody concentration with Nanodrop. (Con. (mg/ml)= A280nm/1.4) Note: The reduced antibody is not stable; the conjugation reaction needs to be carried out the conjugation as soon as possible after purification.

Conjugate with ReadiUse™ Preactivated APC-Cy7 Maleimide

Reconstitute ReadiUse™ Preactivated APC-Cy7 Maleimide in 100 µL ddH₂O to 10 mg/mL. Note: Reconstituted ReadiUse™ Preactivated APC-Cy7 Maleimide are stable at 4 °C for one week, please kept it from light.
Add reduced antibody to pre-activated APC-Cy7 directly to at the ratio of 130 µg APC-Cy7 /100 µg reduced antibody.
Rotate the mixture for 60-120 mins at room temperature.
After 60 minutes, block the free sulfhydryls on the antibody.
Prepare a fresh solution of 10 mg/mL NEM in DMSO; add 3.4 µL per mg of antibody and rotate for 20 minutes at room temperature.

Purification

Antibody/APC-Cy7 conjugate could be further purified through size exclusion chromatography to get best performance. Note: The Antibody/APC-Cy7 conjugate solution is recommended to be stored at 2~8 °C and kept from light.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	779

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)
ReadiUse™ Preactivated APC-Cy7 Tandem	651	779	700000
ReadiUse™ Preactivated PE-Cy7 Maleimide	565	778	1960000
ReadiUse™ Preactivated PerCP-Cy7 Maleimide	482	784	350000

Images

Figure 1. Flow cytometry analysis of whole blood stained with APC-Cy7 anti-human CD8 *HIT8a* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the APC-Cy7 specific R7-A channel.

References

View all 17 references: Citation Explorer

CD4+ T cells and natural killer cells: Biomarkers for hepatic fibrosis in human immunodeficiency virus/hepatitis C virus-coinfected patients.
Authors: Laufer, Natalia and Ojeda, Diego and Polo, María Laura and Martinez, Ana and Pérez, Héctor and Turk, Gabriela and Cahn, Pedro and Zwirner, Norberto Walter and Quarleri, Jorge
Journal: World journal of hepatology (2017): 1073-1080

Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry.
Authors: De Biasi, Sara and Gibellini, Lara and Bianchini, Elena and Nasi, Milena and Pinti, Marcello and Salvioli, Stefano and Cossarizza, Andrea
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2016): 1106-1110

Presence of CD34(+)CD38(-)CD58(-) leukemia-propagating cells at diagnosis identifies patients at high risk of relapse with Ph chromosome-positive ALL after allo-hematopoietic SCT.
Authors: Kong, Y and Xu, L-P and Liu, Y-R and Qin, Y-Z and Sun, Y-Q and Wang, Y and Jiang, H and Jiang, Q and Chen, H and Chang, Y-J and Huang, X-J
Journal: Bone marrow transplantation (2015): 348-53

Analysis of Populations of Memory T-Helper Cells Expressing CXCR3 and CCR6 Chemokine Receptors in Peripheral Blood of Patients with Chronic Viral Hepatitis C.
Authors: Elezov, D S and Kudryavtsev, I V and Arsent'ev, N A and Basin, V V and Esaulenko, E V and Semenov, A V and Totolyan, A A
Journal: Bulletin of experimental biology and medicine (2015): 238-42

A flow cytometric method for the analysis of macrophages in the vascular wall.
Authors: Moore, Jeffrey P and Sakkal, Samy and Bullen, Michelle L and Kemp-Harper, Barbara K and Ricardo, Sharon D and Sobey, Christopher G and Drummond, Grant R
Journal: Journal of immunological methods (2013): 33-43

Combined normal donor and CLL: Single tube ZAP-70 analysis.
Authors: Degheidy, Heba A and Venzon, David J and Farooqui, Mohammed Z H and Abbasi, Fatima and Arthur, Diane C and Wilson, Wyndham H and Wiestner, Adrian and Stetler-Stevenson, M A and Marti, Gerald E
Journal: Cytometry. Part B, Clinical cytometry (2012): 67-77

The role of CD19 and CD27 in the diagnosis of multiple myeloma by flow cytometry: a new statistical model.
Authors: Cannizzo, Elisa and Carulli, Giovanni and Del Vecchio, Luigi and Ottaviano, Virginia and Bellio, Emanuele and Zenari, Ezio and Azzarà, Antonio and Petrini, Mario and Preffer, Frederic
Journal: American journal of clinical pathology (2012): 377-86

Measurement conditions for flow cytometry analyses of cell lines from urological carcinomas.
Authors: Tölle, Angelika and Abdallah, Ziyad and Jung, Klaus and Bäumler, Hans
Journal: Journal of fluorescence (2010): 779-86

Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.
Authors: Le Roy, Christine and Varin-Blank, Nadine and Ajchenbaum-Cymbalista, Florence and Letestu, Rémi
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2009): 882-90

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood.
Authors: Hristov, Mihail and Schmitz, Susanne and Schuhmann, Christoph and Leyendecker, Thorsten and von Hundelshausen, Philipp and Krötz, Florian and Sohn, Hae-Young and Nauwelaers, Frans A and Weber, Christian
Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology (2009): 848-53