Olympus R1 Instructions Manual - Page 24

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Chapter 3 – Protocol Settings

The R1 provides a default protocol that can be used for most common cell lines. Users may create and save up

to 300 unique protocols.

3.1 Protocol Parameters

The R1 protocols have the following modifiable parameters:

Parameter

Dilution Factor

Noise Reduction

Live Cell Sensitivity

Roundness (%)

Min. Cell Size (µm)

Max. Cell Size (µm)

Declustering Level

Dilution Factor: The dilution factor is used to calculate cell concentrations accurately. The default dilution

factor is preset as 1 for the Staining option: Without Trypan Blue and 2 for With Trypan Blue, assuming a 1:1

ratio of trypan blue to cell suspension. Users can modify this value according to the dilution of the original

sample in increments of 1 between 2 to 10 and of 10 between 10 to 100. For users handling high density cells

(e.g. fermented CHO cells), serial dilutions and several counts with appropriately adjusted dilution factors will

be necessary.

Noise Reduction: This option allows for the adjustment of background noise during counting. With more noise

reduction, the instrument will be less sensitive and not detect weakly stained cells. With lower noise reduction,

the instrument can detect objects with faint signals. Adjusting this parameter will help optimize for different cell

types as trypan blue staining can vary from cell to cell.

Live Cell Sensitivity: Live cells with intact cell membranes exclude trypan blue. Trypan blue forms annular

shade around live cells and stain the cytoplasm of dead cells or cells with compromised membranes. With a

higher live cell sensitivity, the instrument can detect smaller cells by registering smaller annular shade. For

most cells, this option should be set to 1. For peripheral blood mononuclear cells (PBMCs) or smaller, set it to ≥

Roundness: As not all cells are completely spherical, adjusting the roundness allows for the detection of a

variety of cells. Higher percentages lead to the counting of rounder cells and excludes objects with less

roundness. Lower percentages are suitable for counting cells with irregular shapes.

Minimum and Maximum Cell Size: Cell sizes also vary. Users can customize cell size parameters to detect

specific cells efficiently. Values can be adjusted in 1 µm increments for sizes between 3-60 µm.

Declustering Level: The declustering function allows for the efficient detection of a variety of cells that may

clump or grow in clusters. Higher levels of declustering will increase counting time. This function is helpful for

counting sticky cells or rod-shaped spores.

Range

1 - 100

1 - 10

1 - 9

0 - 100

3 - 59

4 - 60

None, Low, Medium, High

DEFAULT

Medium