PE-Cy7

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.

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