Protonex™ Red 600-Latex Bead Conjugate

The following is a recommended protocol for granulocytes. This protocol only provides a guideline and should be modified
according to your specific experimental conditions. 

1. Prepare cells as desired. For example, prepare the granulocytes at 10⁷ cells/mL with Hanks and 20 mM Hepes buffer (HHBS), and add 100 μL to a polypropylene tube.

   Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density. 

2. Add 1-10 μL of the Protonex™ Red 600-Latex Bead Conjugate to the tube and incubate with gentle shaking for 30 minutes at 37˚C.

   Note: Each cell line should be evaluated on an individual basis to determine the optimal incubation time. 

3. Prepare an identical sample that is incubated at 4˚C and label it as a control.

4. At the end of the 30-minute incubation, stop phagocytosis by adding 2mL of ice-cold HHBS and mix well.

5. Wash the cells 2 times with cold HBSS. 

6. Resuspend the cells in 500 μL of cold HBSS, keep the samples at 4˚C, and analyze immediately using a fluorescence microscope equipped with a Texas Red® filter set.

   Note: For fluorescence microplate readers, monitor the fluorescence intensity at Ex/Em = 570/600 nm (Cutoff = 585 nm).