Cell Type Guidelines & Optimization

vi-cell blu cell type guidelines

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
    Apsirate 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.