7 aad facs calibur software

Do i need to do any compensation between apc and pi in flow cytometry. As said, pi works fine and 7aad can be an alternative as it is very similar to pi and in theory not excited by the 633 nm laser. How to compensate a 4color flow cytometry experiment. Sytox blue is excited by the 405 nm laser and its emission is far away from apc. Analyse by flow cytometry collecting 25,000 events per sample. To adjust flow cytometer settings for 7aad, add 5 10. Depending on your machine and your combination, you can also use dyes excited by other lasers. It is also one of the areas thats steeped in mystery, myths and misinformation.

Hoechst 33342 titrated for each cell type and has to be incubated with live cells at 37oc. See the bd calibrite beads package insert and the bd facscomp software reference manual for staining and setup instructions. Since the initial commercialization of flow cytometry fc and fluorescence activated cell sorting facs in 1968, they have undergone significant improvements. Do i need to do any compensation between apc and pi in. Optimize your flow cytometry best practices for sample preparation, staining and analysis duration. Bd biosciencesfluorochrome reference chart w d stain index dw resolution sensitivity the ability to resolve a dim positive signal from background is a function of the difference between positive and background peak means d and the spread of the. Pi works fine and 7 aad can be an alternative as it is very similar to pi and in theory not excited by the 633 nm laser. This solution can be used in combination with pe phycoerythrin and fitc fluorescein isothiocyanate conjugated antibodies in 2color analysis. Note you can use either 3color or 4color lnw settings to set up the cytometer. Mix gently and incubate for 30 minutes at 4 c in the dark. Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. Pi is normally used at 50 gml, 7 aad at 25 gml, topro3 10nm, dapi at 1gml. A practical guide for using flow cytometry and cell sorting, including extensive discussion on hardware, suppliers, reagents, and software. Manually adjusting the compensation values based on how the populations look, or socalled cowboy compensation, is not the correct way to determine proper compensation.