Hello Lucian Miles

You may incorporate the following in your protocol.

1. Cell treatment: After digestion, should I use PBS or serum-free medium? What is the appropriate centrifugation speed?

You should use serum-free medium. The appropriate centrifugation speed should be 300 x g for 5 mins.

2. Staining conditions: Is the working concentration of the dye 2 μM? How long should it be incubated at room temperature? Is a light-proof shaker required?

Yes, the final working concentration of the dye is 2μM. You may add the cells to the dye and the sample should be immediately mixed by pipetting because rapid and homogeneous mixing is essential for uniform labeling since the staining is nearly instantaneous. The sample may be incubated at 25°C for 2 to 5 minutes. As you mentioned, a light-proof shaker is recommended for PKH26 cell labeling. The PKH26 dye solution and labeled cells should be protected from bright direct light throughout the staining process to prevent photobleaching and ensure optimal staining.

3. Washing steps: After stopping the reaction and adding 10% serum, should I wash 2 or 3 times to remove excess dye?

Yes. The staining reaction is stopped by adding an equal volume of serum and incubated for 1 min. This serum-stopped sample is diluted with an equal volume of complete medium. The cells are then centrifuged at 300 x g for 10 minutes at 25°C to remove cells from staining solution. The supernatant may be removed and the cell pellet may be transferred to a new tube for further washing (a minimum of 3 washes is recommended). Use complete culture medium to wash the cells and then centrifuge the cells. Resuspend to the desired concentration.

Best,