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Summary of Contents for ChemoMetec NucleoCounter NC-3000 FlexiCyte
- Page 1 Quick Guide ® NucleoCounter ™ NC-3000...
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Page 2: Table Of Contents
Table of contents Setting up the FlexiCyte Protocol Editing Image Capture and Analysis Parameters Optimizing Exposure Time Compensation for Spectral Overlap Creating and Modifying Graphs Gating and Data Analysis Polygons • • Quadrants Markers • Table plots • Identifying the Sub-Population in the Image Window Gating Cell Populations for Inclusion/Exclusion in Analysis Generic Gate... -
Page 3: Setting Up The Flexicyte Protocol
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. -
Page 4: Editing Image Capture And Analysis Parameters
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 •... -
Page 5: Optimizing Exposure Time
UV LED Violet LED Blue LED Green LED Red LED Darkfield (Couterstain) Light source Ex630 Darkfield / Ex365 Ex365 Ex405 Ex475 Ex530 Available emission filters / Em470/55 Em430/20 Em560/35 Em740/60 Em475/15 Em675/75 Em470/55 Em530/15 Em580/25 Em630LP Em475/15 Em560/35 Em675/75 Em740/60 Em560/35 Em675/75 Em630LP... - Page 6 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-3000 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).
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Page 7: Compensation For Spectral Overlap
Compensation for Spectral Overlap Even though fluorophores are designated a particular color, they emit light over a range of wavelengths. The color associated with a particular fluorophore refers to the wavelength where it has maximum fluorescence. However, the wavelengths where it has weaker emission may spill over into a neighboring channel giving a false increase in signal in this channel. - Page 8 Enter an estimated compensation factor into the matrix box associated with the two channels of interest and click ‘Preview’ or use the scale bar for live updated adjustments. Inspect the cell population to ascertain that cell population is spread horizontally across the graph area. If the compen- sation factor is incorrect, click ‘Roll back’, enter a new value and inspect the cell population again.
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Page 9: Creating And Modifying Graphs
Figure A) Scatter plot showing GFP fluorescence in the green and orange channel after compensation. Note, no increase in orange fluorescence is seen as green fluorescence increases. B) Compensation matrix adjusted for 0.8% signal spillover from the GFP into the orange channel. Creating and Modifying Graphs Default graphs representing each of the channels measured in a particular analysis or graphs defined by the master in the protocol are automatically opened in Plot... -
Page 10: Gating And Data Analysis
Gating and Data Analysis Data analysis will often require that debris or sub-populations of cells are identified and removed and statistics for the different populations gathered. Sub-populations of cells can be marked by either quadrants or polygons in scatter plots and markers in histograms. •... -
Page 11: Markers
7. Large scatter Figure plot of a FlexiCyte assay to measure GFP and RFP transfection. Markers • Markers can be inserted on histograms by clicking on the small histogram to open the large histogram in editing mode. Select ‘Create Marker’ and the cursor will change from an arrow to a cross. Create a marker by clicking on the positions in the histogram where the markers are to start and finish. - Page 12 Creating an Overlay of Histograms Histograms from multiple analyses can be displayed in a single graph to allow better visual comparison of the results. Right-click on the small histogram plot to be copied and select ‘Copy Histogram’. Right-click on the small histogram plot to where the histogram is to be pasted and select ‘Paste Histogram’.
- Page 13 Table plots • Add and place a new table in your table plot. (Fig. 9) Figure To fill in text, simply click in the table and write your text To fill in a formula, right-click a table and fill in your calculation a.
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Page 14: Identifying The Sub-Population In The Image Window
Identifying the Sub-Population in the Image Window One of the advantages of image cytometry is that cells defined in a particular sub-population can be identified in the image acquired for analysis. This is done when the image file for the analysis is loaded in the main window and sub-populations have been defined by polygons, quadrants or markers. -
Page 15: Generic Gate
Double-click on the small graph to be analyzed to open the corresponding larger graph in editing mode. Click the ‘Gating Setup’ in the upper left corner to open a list of gating options available for that particular analysis. Gate options are denoted as ‘P’... -
Page 16: Exporting Data To Spreadsheets
Save the row with all adjusted compensations, markers, polygons, quadrants and gatings by clicking on the button in the row dialog. Right-click the file in the file list that represents the data just saved and select ‘Start Protocol Adaptation Wizard’. In the wizard, skip directly to the step ‘Select and setup a master file’, the second last step. -
Page 17: Exporting Data To Other Software
Exporting Data to Other Software Data can be exported in ‘*.acs’ or ‘*.fcs’ formats which are compatible with most third-party cytometry analysis packages. Select the file containing the data to be exported. In the ‘File’ menu select ‘Export Data’. A new window will open allowing you to choose the format to be exported, which parameters are to be exported and the location where the file is to be saved. -
Page 18: Pdf Reports
PDF reports Right-click on file to create a PDF report Figure 10 Select the parameters to be visible on the report and the properties of the parameters Optional: preview your result Save and/or print your report to the default printer... - Page 19 Figure 11 Tip: Batch exports can be done from the NucleoView™ File Browser. Figure 12...
- Page 20 Copyright Notices Copyright © ChemoMetec A/S 2012. All rights reserved. No part of this publication and referred documents may be re- produced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written consent of ChemoMetec A/S, Gydevang 43, DK-3450 Allerod, Denmark.
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