How to setup a FlexiCyte protocol

Background

The NucleoCounter® NC-3000™ system enables the user to perform advanced cell analyses of a broad range of mammalian cells. This FAQ, describes the usage of the FlexiCyte™ protocol that can be modified by the user to perform user defined protocols such as detection and measurement of characteristics of a population of cells or particles by staining cell surface or internal markers. 

How to setup a FlexiCyte protocol

Setup for a 3 color experiment using either Hoechest-33342 or DAPI as Masking

  1. In order to adjust the FlexiCyte™ protocol, select the FlexiCyte™ protocol and open the ‘Protocol Adaptation Wizard’ in the ‘Tools’ menu. 

2. The wizard is a guide for the user to change masking method, the number of analytical channels, light sources, emission filters, channel names and exposure times for the fluorescence channels. 

3. There are 7 steps in the setting up a protocol using the Wizard
a. Step 1. Select the number of Channels to be used, the default is 3 when either Hoechest-33342 or DAPI is used as masking. Click inside number of channels box, it toggles between 1-4. Choose 3 for now.

b. Click on Step 2. This will bring up the Light Source dialog box. By clicking inside the light source boxes, it will toggle between the light sources available. Selection is based on the masking channel at UV (LED365) for Hoechest-33342 or DAPI. Channels are chosen according to the fluorochromes and dyes in the experiment and must fit the excitation for those tags.

c. Click on Step 3 which will bring up the exposure time for each light source. This can stay on the default value. This step requires advance knowledge of the system.

d. Click on Step 4. This will bring up the dialog box for the emission filters, there are 5 filters available for the NC-3000 to choose from. By clicking inside the filter boxes, it will toggle between the filters available. Choose the emission filter available for the fluorochromes or dyes in the experiment. This step also allows you to change the “channel” name to the corresponding fluorochrome or dye in the experiment

e. The protocol is ready to be recalled to run the experiment.

* Step 6 can be setup after either the first sample is run an all the gates and markers have been set or when the experiment is finished. This will enable the data collected at later dates to be displayed correctly.

Before

After

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