ChemoMetec NucleoCounter NC-3000 FlexiCyte Quick Manual

Quick Guide

NucleoCounter

®

NC-3000

™

Table of contents

Setting up the FlexiCyte Protocol 2

Editing Image Capture and Analysis Parameters 3

Optimizing Exposure Time 4

Compensation for Spectral Overlap 6

Creating and Modifying Graphs 8

Gating and Data Analysis 9

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•

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Identifying the Sub-Population in the Image Window 13

Gating Cell Populations for Inclusion/Exclusion

in Analysis 13

Generic Gate 14

Exporting Data to Spreadsheets 15

Exporting Data to Other Software 16

Exporting Graphics 16

PDF reports 17

Polygons

Quadrants

Markers

Table plots

9

10

9

12

1

Setting up the FlexiCyte Protocol

Open the ‘Select Protocol’ window opened with the (Fig. 1A)

Select Organism: Mammalian, and Analysis: FlexiCyte, and Media Type:
NC-Slide A2.

Select the FlexiCyte protocol for setting up a new FlexiCyte protocol (Fig.
1B), or select the protocol that should be modified.

Figure 1.

col (B).

Open ‘Select Protocol window (A), and select the FlexiCyte proto-

2

Editing Image Capture and Analysis Parameters

Open ‘Protocol Adaption Wizard’ (Fig. 2) located under ‘Tools’ on the
main menu bar.

Follow the on-screen help to select:

• The number of analytical channels (see fig. 3 for the available
channels)

• Masking method

• Light sources (LEDs)

• Exposure times

• Emission filters

• Minimum number of cells to analyze

• Whether to include or exclude aggregated cells

A new title must be given to any adapted protocols.

Optional: If a sample has already been run, the wizard can be opened by right
clicking the image file name, and generic data analysis can be set up (See

Template Setup).

Figure 2.

Protocol Adaption Wizard allows the user to define protocols by adjusting
the FlexiCyte™ parameters as described in the help section.

3

Light source

Available emission filters

UV

Darkfield

(Couterstain)

Darkfield / Ex365

- / Em470/55

UV LED

Ex365 Ex405 Ex475 Ex530

Em430/20
Em470/55
Em475/15
Em560/35
Em675/75
Em630LP
Em740/60

Violet LED

Em475/15
Em530/15
Em560/35

Blue LED Green LED

Em560/35
Em580/25
Em675/75

Em675/75
Em630LP
Em740/60

Red LED

Ex630

Em740/60

Figure 3.

A full list of possible LED/Emission filter combinations.
The denotation of emission filter as e.g. Em530/15 means a bandpass filter
that allows light of wavelength 530nm ± 15nm (515nm - 545nm) to pass.

Optimising Exposure Time

Optimizing signal acquisition can easily be performed on the NucleoCounter®
NC-3000TM by adjusting the exposure time in a manner similar to that used in
photography. If the image is under-exposed, it will be darker and much of the
finer detail may not be seen. Similarly, if it is over-exposed the pixels will become
saturated and information will also be lost.

Optimizing the exposure time in the NC-3000TM needs to be determined empirically
for the initial experiment but, once determined, the settings can be applied to
all the following samples. That said, the default setting (200 milliseconds for
LED(365) and LED(405), and 1000 milliseconds for LED(475), LED(530) and
LED(630)) will fit most applications and optimization of exposure time may not
be required.

As an example GFP, coupled to a highly expressed protein, has a significant
chance of being over-exposed with the default of 1000 milliseconds. However
most fluorochrome coupled antibodies bound to an expressed protein will be
appropriately exposed with 1000 milliseconds.

Insert a sample stained with a single fluorophore of interest and masking
stain (DAPI or Hoechst 33342) if flourescent masking is chosen into the
NC-3000.

Set the exposure time to be evaluated in ‘Protocol Adaption Wizard’ (See
section: editing Image Capture and Analysis Parameters).

Run the sample using the protocol with the adjusted exposure time.
The data will automatically open in ‘Plot Manager’. Add a histogram to

data by clicking on the histogram icon on the left-hand side of the
data row.

Double-click on the small histogram to open the large histogram in
editing mode. Change the x-axis to the appropriate channel and the
parameter to ‘Max Intensity’. This will display the signal intensity for the
most intense pixel/cell rather than average intensity for the area defined
as a cell when the scale is set to ‘Intensity’.

4

To evaluate if the exposure time for the channel is appropriate for a particular
channel, examine the distribution of the signal in the histogram. The
NC-3000TM is based on 16-bit imaging that allows acquisition of signals
from approx. 0 - 65,500. If the image is under-exposed ‘normal distribution’
curve may rest against the y-axis and lower intensity events will not be
acquired (Fig 4A). If the image is over-exposed, the maximum intensity
values will be close to 65,500 and a shoulder may be seen on the ‘normal
distribution’ curve (Fig 4B).

If required, adjust the exposure time for your sample as appropriate and
repeat step 2 - 6.

Step 2 - 7 should then be repeated for all channels used in the final assay.
Once the optimal exposure time has been determined for the individual

channels, a final protocol with optimized exposure times for all the channels
is saved in the ‘Protocol Adaption Wizard’.

Figure 4.

Max intensity histograms for A) under-exposed image, B) over­exposed image with a shoulder indicating saturated pixels and C) an
image with correct exposure. Note the smaller scale on the under-exposed
image is different so that the peak can be more readily visualized.

5