Cell Type Guidelines & Optimization

BLU includes 7 system cell types:

• BCI Default – general use
• Mammalian - CHO, HEK, PBMC
• Insect – SF-9, SF-21
• Yeast- biofuel, breweries, fermentation applications
• BCI Viab Beads – for use with BLU viability control
• BCI Conc Beads – for use with BLU concentration controls
• BCI L10 Beads – for use with L10 (10um) size standard

For many cell types, the default cell type values provided in the software are suitable. For some Cell Lines, parameters will need to be changed to optimize the analysis of the cells.

Migrating cell types from XR to BLU

Suggested conversion values. Please contact your Beckman Coulter Life Sciences representative for more assistance.

	XR Setting	BLU Setting Starting Value
Min	Same	-1 um
Max	Same	-1 um
Images	50	50 or 100
Aspirate	Same	3
Mix	Same	3
Cell Brightness	85	N/A
Cell Sharpness	100	7
	80	15
	60	20
Viable Spot Brightness	85	55
	75	50
	65	40
Viable Spot Area	Same	Same
Min Circularity	Same	Same
Decluster	Same	Same

 

	Cell Type Parameters	Typical Range	Full Range	Increase Value	Decrease Value	When/Why You Want to Change This	Warnings
Defines whether there is a cell present or not	Minimum Diameter	5 to 12 µm	1 to 60 µm	Less cells identified	More cells identified	Software is excluding small cells (circled blue) Debris included in analysis	Too low, may be including debris Too high, cells excluded
	Maximum Diameter	15 to 50 µm	1 to 60 µm	More cells identified	Less cells identified	Software is excluding large cells (circled blue) Clumps of cells are included	Too low, may be excluding cells Clumps of cells included
	Cell Sharpness	5 to 25	0 to 100	Less cells identified	More cells identified	Software is excluding "fuzzy" cells Unwanted debris is being picked up	Set too high may cause dead cell to be excluded and alter the percent viability.
Defines whether identified cells are viable or dead	Viable Cell Spot Brightness	40 to 60%	0 to 95%	More cells identified as dead	More cells identified as viable	Viable cells are identified as dead Dead cells are identified as viable	Too low, may start identifying dead cells as live Too high, may start identifying live cells as dead
	Viable Cell Spot Area	3 to 12%	0 to 95%	More cells identified as dead	More cells identified as viable	Viable cells are identified as dead Dead cells are identified as viable	Too low, may start identifying dead cells as live Too high, may start identifying live cells as dead
Defines whether there is a cell present or not	Minimum Circularity	0 to 0.60	0 to 1.00	More irregular shaped dead cells will be excluded	Less irregular shaped dead cells will be excluded	Unwanted irregular shaped debris are being picked up	Too low, may be capturing unwanted debris Too high, may start excluding cells
	Decluster Degree	None, Low to Medium	N/A	More cells identified	Less cells identified	Single cells are being identified as 2 or more cells	Too low, may be missing cells in clusters Too high, may over decluster by annotating single cells as 2 or more cells
Cell Sharpness	Cell sharpness is a measure of cell edges ranging from 0-100. Decreasing the value allows for fuzzier objects to be identified as cells. Increasing the sharpness will eliminates fuzzier cells from the cell count.
Viable Cell Spot Brightness	Viable Cell Spot Brightness measures the brightness of the center of the cells to determine whether the cell is viable or dead (green or red annotation). At high values cells with bright white centers are counted as viable (viability will decrease). At low values cells with grey centers will be counted as viable (viability will increase). It is important to check annotation to determine if Viable Cell Spot Brightness is set correctly.
Viable Cell Spot Area	Viable Cell Spot Area is a percentage of the white area of a cell used to determine if the cell is viable or dead. A high value may cause some viable cells to be counted as dead. It is normally not necessary to adjust this parameter.
Minimum Circularity	This parameter is used to eliminate debris. Assume "1" is a perfect circle.
Decluster Degree	The declustering degree defines intensity of declustering clumps of cells. None will not decluster any clumps of cells - Good for samples with cells that are very long and contain no cell clumps. Low is good for samples with clumps of 2-3 cells. Medium is good for samples with some clumps of with more than 3 cells. High is good for yeast cells.

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