CY5-Dextran (MW60-80k)

The maximum excitation wavelength of CY5 is 633 nm, and its maximum emission wavelength is 670 nm.
Guidelines (The following are recommended experimental protocols for guidance only; adjust according to actual requirements)

I. Stock Solution Preparation

1. Cy5-dextran Conjugation (Homemade): Weigh 10 mg dextran (70 kDa, within the range of 60-80 kDa) and dissolve it in 2.4 mL of 0.1 M sodium bicarbonate buffer (pH 9.5). Separately, dissolve 1.0 mg Cy5.18-OSu (Cy5 active ester) in 140 μl anhydrous dimethylformamide (DMF). Add 100 μl of the Cy5 solution to the dextran solution, mix vigorously, and incubate at room temperature in the dark for 1 h. Remove free Cy5 from the reaction mixture using a Sephadex G-25 gel chromatography column, collect the labeled product, dialyze thoroughly against Tris-buffered saline (TBS, pH 7.4) followed by PBS, aliquot, and store at -20°C in the dark^[1][2][3].
2. Commercial FITC-dextran Stock Solution: Prepare FITC-dextran (Sigma, FD70, 70 kDa) in PBS at a concentration of 50 mg/mL, then dialyze sequentially against TBS (pH 7.4) and PBS before use^[1].
3. Control Drug Stock Solutions: Prepare Bafilomycin A1 in DMSO at 20 mM and store at -20°C; prepare Nocodazole in DMSO at 6 mg/mL (approximately 20 mM) and store at -20°C. The final concentration of DMSO in working solutions should be <1%^[1][2].

II. Working Solution Preparation

1. Flow Cytometry for Endosomal pH (Suspension Cells): Add FITC-dextran to pre-warmed MEM (containing 10% FCS) to a final concentration of 6 mg/mL, and Cy5-dextran to a final concentration of 1 mg/mL. Prepare fresh before use^[1][2].
2. Virus Co-internalization Assay (FACS/SOFA): Add FITC-dextran at 6 mg/mL + Cy5-dextran at 0.6 mg/mL to MEM (containing 10% FCS), and simultaneously add adenovirus (1,000 particles/cell) or HRV2 (1,000 TCID₅₀/cell)^[3].
3. Fluorescence Microscopy (Adherent Cells): Add FITC-dextran at 10 mg/mL + Alexa568-transferrin at 25 μg/mL to pre-warmed serum-free MEM^[1].
4. In Vivo Animal Imaging: Prepare Cy5-dextran in PBS at 10 μg/mL (for tail artery or local gland injection); or use it in combination with TXR-Tfn (10 μg/mL).

III. Staining Procedures

Scenario A: Suspension Cell Pulse-Chase (pH Measurement by Flow Cytometry)
- Culture HeLa Ohio cells in suspension to a density of 2×10⁵/mL, take 2×10⁷ cells, and pre-incubate them in serum-free MEM at 37°C for 30 min (if drugs are added, maintain 200 nM Bafilomycin A1 or 20 μM Nocodazole throughout the process)^[1][2].
- Collect cells by centrifugation, resuspend them in 2 mL of pre-warmed MEM containing 6 mg/mL FITC-dextran + 1 mg/mL Cy5-dextran, and perform pulse labeling at 37°C for 5 min^[1][2].
- Add pre-chilled PBS (pH 7.4) to end endocytosis, wash twice with PBS, resuspend in 2 mL of unlabeled MEM, and perform chase incubation at 37°C for specified time points such as 0, 15, and 30 min^[1][2].
- Take 100 μl of cell suspension at each time point, add ice-cold PBS to terminate the reaction, and analyze immediately by flow cytometry^[1][2].

Scenario B: Adherent Cell Pulse-Chase (Fluorescence Microscopy)
- Seed HeLa cells onto coverslips and culture overnight until semi-confluent; pre-incubate in serum-free MEM at 37°C for 30 min to deplete endogenous transferrin^[1].
- Add 200 μl of pre-warmed MEM (containing 25 μg/mL Alexa568-transferrin + 10 mg/mL FITC-dextran) and perform pulse labeling at 37°C for 5 min^[1].
- Wash three times with pre-warmed MEM, add 500 μl of unlabeled MEM, and perform chase incubation at 37°C for 15, 35, and 55 min^[1].
- Wash with ice-cold PBS++ (PBS containing 1 mM CaCl₂ + 1 mM MgCl₂); incubate with acid wash buffer (150 mM NaCl, adjusted to pH 3.0 with glacial acetic acid) at 4°C for 2×5 min to remove plasma membrane-bound transferrin; incubate with PBS++ (pH 7.4) at 4°C for 2×5 min to release apotransferrin^[1].
- Fix with 4% paraformaldehyde for 1 h at room temperature, quench with 50 mM NH₄Cl, and mount with Moviol^[1].

Scenario C: In Vivo Rat Salivary Gland Injection
- Anesthetize Sprague-Dawley rats (150-250 g) via intramuscular injection of ketamine (100 mg/kg) + xylazine (20 mg/kg).
- Expose the submandibular glands, and inject 50 μl of Cy5-dextran (10 μg/mL) into the lateral side of each gland; alternatively, inject a mixed probe (10 μg/mL TXR-Tfn + 70 kDa Alexa488-dextran) via the tail artery.
- Harvest the tissues 1 h after injection and place them in ice-cold 1% BSA/DMEM; or directly perform in vivo two-photon microscopy time-lapse imaging.

Scenario D: Primary Stromal Cell Isolation and Relabeling
- Harvest the glands 1 h after Cy5-dextran injection, mince them, and digest with 2 mg/mL collagenase + 0.125 mg/mL dispase at 37°C for 1 h; pipette to disperse the cells, filter through sterile gauze, and centrifuge at 2,000 rpm for 5 min.
- Seed the cells onto glass coverslips, culture at 37°C with 5% CO₂ for 24 h; after incubating in 0.5% BSA/DMEM for 30 min, perform relabeling with TXR-Tfn (10 μg/mL) + Alexa488-dextran (70 kDa).

IV. Detection and Analysis

1. Flow Cytometry (FACS) Parameters
- Instrument: Dual-laser FACS Calibur (BD) equipped with an argon ion laser and a red diode laser^[1][3].
- FITC channel: Excitation at 488 nm, emission collected with a 530 nm bandpass filter (30 nm bandwidth)^[1][3].
- Cy5 channel: Excitation at 635 nm, emission collected with a 661 nm bandpass filter (16 nm bandwidth)^[1][3].
- Collect FSC, SSC, and dual fluorescence signals simultaneously with 256-channel resolution; collect 10,000 cells per sample for cell suspension analysis, and 100,000 events per sample for single-organelle flow analysis (SOFA), with 8 replicate measurements per sample^[1][3].

2. pH Calibration Curve
- Aliquot the labeled cells into 6-8 equal parts, and resuspend each part in calibration buffers with pH values of 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 (prepared by mixing 50 mM HEPES and 50 mM MES in appropriate ratios, containing 50 mM NaCl, 30 mM ammonium acetate, and 40 mM sodium azide)^[1][3].
- Add Monensin to dextran samples to a final concentration of approximately 0.67 μM (by adding 1 μl of 200 μM stock solution to 300 μl of sample), and place at 4°C for 5-10 min to deplete ATP and equilibrate intravesicular pH^[1].
- Measure the mean fluorescence intensities of FITC and Cy5 at each pH, subtract the autofluorescence of unlabeled cells; calculate the FITC/Cy5 fluorescence ratio, normalize the ratio at pH 7.4 to 1.0, and plot the pH standard curve^[1][3].

3. Result Interpretation
- Cy5 fluorescence serves as a pH-insensitive internal reference, while FITC fluorescence quenches less as pH decreases (acidification); the FITC/Cy5 ratio is positively correlated with endosomal pH, and substituting the sample ratio into the calibration curve yields the average endosomal pH^[1][3].
- Normal endocytic pathway: After 5 min of pulse labeling, dextran localizes to peripheral early endosomes (pH ~6.0-6.2), and gradually translocates to perinuclear late endosomes/lysosomes (pH ~5.5-4.5) during the chase period^[1][2].
- Viral escape detection: When adenovirus causes endosomal lysis, 70 kDa FITC/Cy5-dextran is released from acidic endosomes into the neutral cytoplasm, leading to an increased FITC/Cy5 ratio and a ~40% reduction in the number of fluorescent endosomes in SOFA; HRV2 only forms limited pores (~10 ?), allowing 10 kDa dextran to leak out while 70 kDa dextran remains trapped in endosomes, where the pH maintains a low value^[3].
- Drug effects: 200 nM Bafilomycin A1 blocks V-ATPase, raising endosomal pH to neutral and trapping dextran in early endosomes; 20 μM Nocodazole disrupts microtubules, causing dextran to accumulate in endosomal carrier vesicles (ECV) and blocking its transport to late endosomes^[1][2].

4. Fluorescence Microscopy Detection
- Multi-band filter set (Zeiss): FITC excitation filter F84-490, Alexa568 excitation filter F83-570, three-color emission filter F83-101; maintain identical exposure time and image processing parameters across all experimental groups^[1].
- Two-photon in vivo imaging: Excitation wavelength of 930 nm for Cy5-dextran, and 840 nm for Alexa488-dextran/TXR-Tfn; use a 60× water-immersion objective (NA 1.2) with an acquisition speed of 0.3 frames/second.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.