CytoWatch™ Wash-free fluorescence cell imaging buffer *10X*

Thaw 10X CytoWatch™ Wash-free fluorescence cell imaging buffer at room temperature before use.

Note: It is OK to use if the buffer has precipitates.

Prepare a 1X CytoWatch™ Wash-free fluorescence cell imaging buffer by adding 1 mL of 10X CytoWatch™ Wash-free fluorescence cell imaging buffer to 9 mL of HHBS (1X Hank’s with 20 mM Hepes buffer, pH 7.0, cat#20001) or a buffer of your choice, and mix well.

Note: 10 mL of 1X staining buffer is enough for one plate. The buffer is stable at room temperature. It is recommended to aliquot and store any unused 10X assay buffer at < -20 °C. Protect from light. Avoid repeated freeze-thaw cycles.

Add the cell staining dye stock solution (generally, a concentrated DMSO solution) into 1X CytoWatch™ Wash-free fluorescence cell imaging buffer (from Step 2) to make the final well concentration 2X of the desired concentration.

Add the 2X Assay Solution (from Step 3) to the microplate well, making sure it's the same volume as the cell culture medium (e.g., 100 µL/well/96-well or 25 μL/well/384-well).

Incubate the cells in a 37 °C, 5% CO2 incubator, or as desired.

Note: The staining dye has the potential to disrupt the effectiveness of the 1X CytoWatch™ Wash-free fluorescence cell imaging buffer. If this occurs, it is advisable to utilize a preferred cell staining method and swap out the cell staining solution with either the cell growth medium or HHBS. Following this, add 100 µL/well/96-well (25 μL/well/ 384-well) of 1X CytoWatch™ Wash-free fluorescence cell imaging buffer into each respective well.

Observe the cells with a fluorescence microscope or a plate reader as required.