FSC-A Scaling

FSC-A Scaling

Area and Height of FSC pulses must be the same. 45

WHY?

The FSC threshold value is based on the FSC pulse height. NOT area The value of 5000 is not relevant on the FSC-A axis since FSC-A FSC-H by default

WHY does FSC-A FSC-H? The laser is only 8 um high and the cell is larger than the 8 um EXCEPTION, these cells are not larger than 8um: bacteria, yeast and RBC’s or platelets

B C signal The pulse flattens out on top because the cell is larger than the laser. Without being fully illuminated the FSC-H pulse can never reach its true maximum FSC-A will always be equal to the full FSC value of the cell, therefore FSC-A FSCH IF the cell is larger than the laser Series1 A D time D laser A B C

You must multiply the FSC-A by some number less than 1 to make the FSC-A FSC-H

HOW?

Adjust the FSC-A Scaling number so the cells fall on a 45 angle on a FSC-A vs. FSC-H plot

BEFORE FSC Area Scaling adjustment AFTER FSC Area Scaling adjustment FSC Area Scaling 0.75 FSC Area Scaling 0.42

Now the threshold value of 5000 has relevance on a FSC-A vs. SSC-A plot debris P1 live cells dead cells

In conclusion FSC threshold is based on the FSC-H Adjust the FSC-A using the FSC-A Scaling under cytometer window laser tab So FSC-A FSC-H by placing the cells on a 45 angle on a FSC-A vs. FSC-H plot