When performing a calcium flux assay in living cells do probes such as Fluo 8, Cal 520 or Calbryte 520 require a washing step after incubation, similar to that of Fluo-4?

In general, yes, you will use the same protocol for Fluo 8, Cal 520 or Calbryte 520 as you would for Fluo-4, especially if you are using Fluo-8. However, here are some reference tips to keep in mind when using calcium dyes:

1. One can perform dye-loading in a growth medium (so cells are much healthier). Then, instead of washing, replace the dye loading medium with Hanks and 20 mM Hepes buffer (HHBS) before performing your calcium assay. If the cells you are using contain anion transporters such as CHO cells, you will need to add probenecid to the dye loading buffer, as well as the HHBS buffer.
2. Compared to Fluo-4 and Fluo 8, Cal-520 AM has a much better cellular retention and signal-to-background ratio. For this reason, you can use less or no probenecid for cells that contain anion transporters to reduce any toxic effects caused by probenecid.
3. Calbryte 520 AM is a significant upgrade to our leading Cal 520 dyes. Compared to other commercially available calcium dyes, Calbryte 520 AM has a significantly brighter signal when used under the same conditions. Like Fluo-8, Calbryte 520 AM has a quicker loading time as it can be done at room temperature. And similar to Cal 520, Calbryte 520 has excellent cellular retention which eliminates the use of probenecid.
4. If you are performing HTS or HCT, we recommend using our NO wash assay kit such as Screen Quest Calbryte 520 Probenecid Free and Wash-Free Calcium Assay Kit (Cat# 36318)