Protonex™ Green 500 Dextran

1. Prepare cells in a growth medium
2. Replace the medium with Protonex™ Green Dextran loading solution (100 µL/well for 96-well plate)
3. Incubate at 37ºC for 5–20 minutes
4. Read Fluorescence at Ex/Em = 443/505 nm

For guidelines on cell sample preparation, please visit:
https://www.aatbio.com/resources/guides/cell-samplepreparation.html

The following protocol is recommended for standard cell loading. It serves as a general guideline and may be adapted to suit specific experimental requirements.

1. For example, plate adherent cells overnight in growth medium at 40,000 to 80,000 cells/well/100µL for a 96-well plate or 10,000 to 20,000 cells/well/25µL for 384-well plates.
   
   Note: Optimal cell density may vary by cell line and should be determined experimentally.

1. Prepare a 1mg/mL stock solution of Protonex™ Green Dextran in 1 mL of sterile water or Hanks and 20 mM Hepes buffer
   (HHBS). The stock solution should be used promptly. Any unused solution need to be aliquoted and refrozen at < -20 oC.
   
   Note: Avoid repeated freeze-thaw cycles, and protect from light.
2. Prepare a 20-100ug/mL Protonex™ Green Dextran loading solution in HHBS.

1. Remove the medium, and add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) Protonex™ Green Dextran loading
   solution into the cell plate (from Step 2.2).
   
   Note: It is important to replace the growth medium with HHBS buffer (100 µL/well for 96-well plate or 25 µL/well for 384-
   well plate before dye-loading) if your compounds interfere with the serum.
   
   Note: Rapid trafficking of Protonex™ Green dextran from early endosomes to late endosomes and subsequent fusion with
   lysosomes can occur. To aid the visualization of Protonex™ Green dextran within the endosomes, we recommend increasing
   the labeling concentration and decreasing the loading time, and imaging immediately.
2. Incubate the dye-loading plate at cell incubator for 5 to 20 minutes.
3. Wash and replace the dye-loading solution with HHBS or growth medium.
4. Run the endocytosis assay by monitoring the fluorescence at Ex/Em = 443/505 nm.

Notes:

• Protonex™ Green Dextran becomes bright green under acidic conditions (e.g., in lysosomes), enabling monitoring of endocytosis and lysosomal acidification.
• The fluorescence signal is relatively stable for at least one hour after trafficking to lysosomes.
• Because lysosomes have a lower pH than endosomes, lysosomal staining is typically brighter. Modulation of endocytosis or lysosomal function can be inferred by changes in fluorescence intensity.