PE-Cy7 is a tandem dye conjugate based on fluorescence resonance energy transfer (FRET). Composed of phycoerythrin (PE) as the donor and Cy7 as the acceptor fluorescent dye, PE-Cy7 is suitable for multicolor flow cytometry, laser scanning cytometry and immunofluorescence applications. PE-Cy7 can be conjugated to antibodies or used in the biotin-streptavidin system to label cell surface antigens in immunophenotyping and multiparametric cell analysis. PE-Cy7 undergoes photon-induced oxidative degradation, whose rate depends on light intensity and storage conditions, leading to reduced FRET efficiency and increased PE leakage. PE-Cy7 can be excited at wavelengths of 488 nm, 532 nm or ~760 nm, and its emission signal can be detected using 780/60 nm, 810/90 nm or 785/50 nm bandpass filters, with a Stokes shift of 300 nm [1] [2] [3].
In Vitro, Operating Instructions (The following is a recommended procedure for reference only, and adjustments are required based on specific requirements) 1. Stock Solution Preparation 1.1 Solvents: 1.1.1 Storage buffer containing 0.016 M phosphate buffer (pH 7.4), 0.02% sodium azide and 0.2% bovine serum albumin. 1.1.2 Anhydrous DMSO. 1.2 Concentration Recommendations: 1.2.1 The concentration of bis-Cy7 stock solution is 10 mg/mL (10 mM). 1.2.2 Note: No stock solution concentration is specified for PE-Cy7 supplied as a conjugated antibody/streptavidin conjugate. 2. Working Solution Preparation 2.1 Diluent: 2.1.1 Dilute PE-Cy7-conjugated streptavidin or antibody at a ratio of 1:10 before use. 2.1.2 Note: PE-Cy7 supplied as a conjugated antibody/streptavidin conjugate requires no dilution and can be used directly. 2.2 Working Concentration: Optimize the concentration via titration to achieve saturated staining. 2.3 Note: Adjust the working solution concentration as needed; prepare and use it immediately. 3. Staining Procedure 3.1 Sample Types: Suitable for suspension cells (human peripheral blood mononuclear cell fractions, human peripheral blood leukocytes, peripheral blood mononuclear cells (PBMC))[1][2][3]: 3.1.1 If whole blood leukocytes are used, process EDTA-anticoagulated blood samples. 3.1.2 Use at least 106 cells per stained sample. 3.1.3 Wash the cells twice before staining. 3.2 Incubation Conditions: 3.2.1 Direct staining: Incubate cells with PE-Cy7-conjugated antibody for 15 min at room temperature in the dark, or for 15 min at 0℃ (ice bath); optimize conditions as needed. 3.2.2 Indirect staining: Aliquot blood samples and incubate with biotinylated primary antibody for 15 min at room temperature, then add PE-Cy7-conjugated streptavidin diluted at 1:10, and continue incubation for 15 min at room temperature. 3.3 Washing Steps: 3.3.1 After staining is completed, lyse red blood cells, then wash the samples with phosphate buffer. 3.3.2 Wash three times with staining medium (RPMI medium without biotin and flavins, supplemented with 4% fetal bovine serum and 0.02% sodium azide). 4. Control Setup 4.1 Set up unstained control, single-positive control, and Full-Minus-One control samples. 4.2 Positive controls are used to validate the experimental system; blank controls are used to eliminate reagent fluorescence interference. 5. Detection and Analysis 5.1 Instrument Types: Flow cytometer, laser scanning cytometer. 5.2 Excitation/Emission Wavelengths: 5.2.1 Excitation light: 532 nm laser, 488 nm argon ion laser. 5.2.2 Emission light: Detect via a 780/60 bandpass filter, detect via an 810/90 nm bandpass filter, or collect emission light >780 nm via a 50 nm bandpass filter with a central wavelength of 785 nm. 5.3 Result Analysis: 5.3.1 Changes in Fluorescence Intensity: 5.3.1.1 Degradation of PE-Cy7 leads to increased PE leakage (the ratio of donor dye emission to acceptor dye emission increases). 5.3.1.2 The PE-Cy7 fluorescence intensity of target-positive cell populations (e.g., CD3-positive lymphocytes) is significantly higher than that of target-negative cell populations. 5.3.1.3 Before reaching the threshold, Cy7 fluorescence intensity increases with the rise of the molar ratio of Cy7 to PE; beyond the threshold, self-quenching causes a decrease in Cy7 fluorescence intensity; meanwhile, due to enhanced resonance energy transfer, PE fluorescence intensity decreases with the rise of the molar ratio of Cy7 to PE. 5.3.2 Fluorescence Localization: Targets to cell surface antigens via conjugated antibodies. 5.3.3 Other Parameters: 5.3.3.1 The resolution parameter of PE-Cy7 for distinguishing positive and negative cell populations is comparable to that of FITC and PE-Cy5. 5.3.3.2 Significant fluorescence spillover occurs into PE and PE-Cy5 channels; minimal spillover occurs into APC and APC-Cy7 channels. 5.3.3.3 Emits far-red fluorescence. 5.3.3.4 Instrument Settings (for laser scanning cytometer): Set the photomultiplier tube gain to 255, the offset to 2075, and the photomultiplier tube voltage to 55% of the maximum voltage.
References:
[1]. Hulspas R, et al. Flow cytometry and the stability of phycoerythrin-tandem dye conjugates. Cytometry. Part A: the journal of the International Society for Analytical Cytology. 2009 Nov;75(11):966-72.
[2]. Gerstner AO, et al. Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry. Cytometry. 2002 Jul 01;48(3):115-23.
[3]. Roederer M, et al. Cy7PE and Cy7APC: bright new probes for immunofluorescence. Cytometry. 1996 Jul 01;24(3):191-7.
In Vitro, Operating Instructions (The following is a recommended procedure for reference only, and adjustments are required based on specific requirements) 1. Stock Solution Preparation 1.1 Solvents: 1.1.1 Storage buffer containing 0.016 M phosphate buffer (pH 7.4), 0.02% sodium azide and 0.2% bovine serum albumin. 1.1.2 Anhydrous DMSO. 1.2 Concentration Recommendations: 1.2.1 The concentration of bis-Cy7 stock solution is 10 mg/mL (10 mM). 1.2.2 Note: No stock solution concentration is specified for PE-Cy7 supplied as a conjugated antibody/streptavidin conjugate. 2. Working Solution Preparation 2.1 Diluent: 2.1.1 Dilute PE-Cy7-conjugated streptavidin or antibody at a ratio of 1:10 before use. 2.1.2 Note: PE-Cy7 supplied as a conjugated antibody/streptavidin conjugate requires no dilution and can be used directly. 2.2 Working Concentration: Optimize the concentration via titration to achieve saturated staining. 2.3 Note: Adjust the working solution concentration as needed; prepare and use it immediately. 3. Staining Procedure 3.1 Sample Types: Suitable for suspension cells (human peripheral blood mononuclear cell fractions, human peripheral blood leukocytes, peripheral blood mononuclear cells (PBMC))[1][2][3]: 3.1.1 If whole blood leukocytes are used, process EDTA-anticoagulated blood samples. 3.1.2 Use at least 106 cells per stained sample. 3.1.3 Wash the cells twice before staining. 3.2 Incubation Conditions: 3.2.1 Direct staining: Incubate cells with PE-Cy7-conjugated antibody for 15 min at room temperature in the dark, or for 15 min at 0℃ (ice bath); optimize conditions as needed. 3.2.2 Indirect staining: Aliquot blood samples and incubate with biotinylated primary antibody for 15 min at room temperature, then add PE-Cy7-conjugated streptavidin diluted at 1:10, and continue incubation for 15 min at room temperature. 3.3 Washing Steps: 3.3.1 After staining is completed, lyse red blood cells, then wash the samples with phosphate buffer. 3.3.2 Wash three times with staining medium (RPMI medium without biotin and flavins, supplemented with 4% fetal bovine serum and 0.02% sodium azide). 4. Control Setup 4.1 Set up unstained control, single-positive control, and Full-Minus-One control samples. 4.2 Positive controls are used to validate the experimental system; blank controls are used to eliminate reagent fluorescence interference. 5. Detection and Analysis 5.1 Instrument Types: Flow cytometer, laser scanning cytometer. 5.2 Excitation/Emission Wavelengths: 5.2.1 Excitation light: 532 nm laser, 488 nm argon ion laser. 5.2.2 Emission light: Detect via a 780/60 bandpass filter, detect via an 810/90 nm bandpass filter, or collect emission light >780 nm via a 50 nm bandpass filter with a central wavelength of 785 nm. 5.3 Result Analysis: 5.3.1 Changes in Fluorescence Intensity: 5.3.1.1 Degradation of PE-Cy7 leads to increased PE leakage (the ratio of donor dye emission to acceptor dye emission increases). 5.3.1.2 The PE-Cy7 fluorescence intensity of target-positive cell populations (e.g., CD3-positive lymphocytes) is significantly higher than that of target-negative cell populations. 5.3.1.3 Before reaching the threshold, Cy7 fluorescence intensity increases with the rise of the molar ratio of Cy7 to PE; beyond the threshold, self-quenching causes a decrease in Cy7 fluorescence intensity; meanwhile, due to enhanced resonance energy transfer, PE fluorescence intensity decreases with the rise of the molar ratio of Cy7 to PE. 5.3.2 Fluorescence Localization: Targets to cell surface antigens via conjugated antibodies. 5.3.3 Other Parameters: 5.3.3.1 The resolution parameter of PE-Cy7 for distinguishing positive and negative cell populations is comparable to that of FITC and PE-Cy5. 5.3.3.2 Significant fluorescence spillover occurs into PE and PE-Cy5 channels; minimal spillover occurs into APC and APC-Cy7 channels. 5.3.3.3 Emits far-red fluorescence. 5.3.3.4 Instrument Settings (for laser scanning cytometer): Set the photomultiplier tube gain to 255, the offset to 2075, and the photomultiplier tube voltage to 55% of the maximum voltage.
References:
[1]. Hulspas R, et al. Flow cytometry and the stability of phycoerythrin-tandem dye conjugates. Cytometry. Part A: the journal of the International Society for Analytical Cytology. 2009 Nov;75(11):966-72.
[2]. Gerstner AO, et al. Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry. Cytometry. 2002 Jul 01;48(3):115-23.
[3]. Roederer M, et al. Cy7PE and Cy7APC: bright new probes for immunofluorescence. Cytometry. 1996 Jul 01;24(3):191-7.