To display the Unit Control or Sample Information control panel, click the title bar.
Sample Information
Original Sample Data
• Displays the sample #. This number defaults to 1 and advances at the completion of sample acquisition.
• Displays the Sample ID for the individual sample.
• Displays the number of events to acquire. The default is 5000. • Displays the dilution factor and the original sample volume. The
default values are 1 and 10, respectively.
• The progress bar provides an estimate of the target event count during acquisition.
Cell Count
Displays the total number of cells and cells/µL that have exceeded the threshold.
Flow Information
Displays the sample flow rate, volume of sample acquired, and acquisition duration.
Threshold Parameter and Threshold Value allow you to select the threshold parameter and enter its value. Click Set after entering the Threshold Value.
Area/Width Parameter allows you to save area and width measurements for a selected parameter.
Set Time Scale allows you to adjust the time parameter axis scale. Click Change Parameter Names to enter your own parameter names (for example, the name of the monoclonal Ab you use). See “Changing Parameter Names” on page 6-26.
Count Gate allows you to select a gate used as a counting gate. All events above the threshold are saved to the file whether they are in the gate or not. However, the number of Events to Acquire is applied to events that fall within the gate.
Sample List Navigation
Allows you to select the previous or next sample from the Analysis Sample List during a data set analysis.
Changing Parameter Names
You can change the default long (or stain) name of any parameter. The short name, for example, GRN-HLog, will still appear in parentheses after the new long name.
1 Click Change Parameter Names in the Sample Information control panel.
The Customize Parameter Names dialog box appears. Area and width appear only if they were selected during the adjust settings step.
2 Highlight the existing name in the Long or Stain Name text field and type in the new name.
3 Click Update.
• You can change the long or stain names for individual samples.
• If you change the names before starting acquisition, the new names will apply to all samples in the run.
• If you use the Apply Current Settings to Selected Samples button during analysis, any changes made to long names will be applied to the selected samples.
Unit Control
guava ExpressPro Assay Troubleshooting
DetectionDisplays the laser status, the FSC, SSC, GRN, YLW, RED, NIR, RED2, and NIR2 gain settings. Displays the area and width scaling factor.
NOTE: Do not change the gains from this panel. Use the
sliders in Adjust Settings to adjust the SSC, GRN, YLW, RED, NIR, RED2, and NIR2 gains.Pump Status
Displays the current status of the pump.
Pump Action
Indicates the current pump position.
Threshold Parameters
Displays the offset and threshold settings for the threshold parameter.
Sample List Navigation
Allows you to select the previous or next sample from the Analysis Sample List during a data set analysis.
Problem Possible Cause Solutions
Message:
This file already exists. You must pick a new name.
Spreadsheet file with same file name already exists in selected directory.
Save guava ExpressPro spreadsheet file to another directory or give it a new name.
Message:
This file exists with read- only attributes. Please use a different file name.
FCS file with same file name already exists in selected directory.
Save guava ExpressPro FCS file to another directory or give it a new name. ExpressPro Software
Module starts in Analysis mode. Acquisition mode is not available.
A registration code was not entered or was entered incorrectly.
Enter the registration code. The code is case sensitive.
Few events, as indicated in Cell Count section of Sample Information control panel.
1. Clogged flow cell.
2. Insufficient sample volume.
3. Cells in suspension have settled.
1. Perform a Backflush. Follow with Quick Clean. 2. Minimum sample volume is
100 µL for round-bottom wells, 150 µL for 0.5-mL tubes, and 900 µL for 1.5-mL tubes.
3. Ensure sample mixing option was selected in WorkEdit Software.
No events, as indicated in Particle Count section of Sample Information control panel.
1. Sample tube or plate not loaded.
2. Insufficient sample volume.
3. Clogged flow cell. 4. Broken flow cell.
5. Sample pump not working. 6. Laser not operational. 7. Loose fitting on minstac
tubing (under metal plate).
1. Ensure tube or plate is in place and tray is loaded. 2. Minimum sample volume is
100 µL for round-bottom wells, 150 µL for 0.5-mL tubes, and 900 µL for 1.5-mL tubes.
3. Perform a Backflush. Follow with Quick Clean. 4. Remove flow cell and
inspect for damage. Replace if necessary. 5. Run Quick Clean and
watch for fluid in waste vial. 6. Contact EMD Millipore
Technical Support.
7. Ensure tubing connector is secure.
Unexpected events appearing in plots displaying GRN, YLW, RED, NIR, RED2, and/or NIR2.
1. Laser not warmed up. 2. Instrument settings not
optimal. Acquiring debris.
1. Allow laser to warm up 10 min before acquisition. 2. Adjust settings so debris is
below threshold. FSC Count under Cell
Count shows events, but the events appear in the wrong places in plots displaying GRN, YLW, RED, NIR, RED2, and/or NIR2.
1. Sample was not stained.
2. Cell lysis.
1. Check sample. If
necessary, restain sample from original suspension. 2. Check buffers used to
process cells.
Events appear in some plots but not in others.
Ensure correct gate is selected for plot in question.
1. Open plot menu, point to Apply Gates and select gate.
2. Check gate definition to ensure it includes the correct regions and operators.
Events appear off scale in dot plots or histograms.
FSC, SSC, GRN, YLW, RED, NIR, RED2, and/or NIR2 gains set incorrectly, or samples staining brightly.
Adjust gain setting so positive populations appear on scale. Repeat Adjust Settings with negative sample. Adjust
compensation settings. Poor resolution between
positive and negative populations.
1. Gains too low to detect fluorescent signals. 2. Incomplete staining with
fluorescent probe, or fluorescent probe
inappropriate for cell type. 3. Fluorescent probes over-
exposed to light, stored improperly, or expired.
4. Non-specific binding of fluorescent probes.
5. Background noise too high.
1. Adjust settings to increase fluorescent signal. Adjust compensation settings. 2. Ensure positive control is
staining adequately and with correct reagent. 3. Refer to reagent package
insert for proper storage instructions. Do not expose reagent to excessive light. Do not use expired
reagents.
4. If using antibody-based probes, try Fc blocking reagent during staining to minimize non-specific binding. Otherwise, titer the fluorescent probes down to reduce the nonspecific staining. 5. Adjust settings to increase
FSC threshold to remove debris. Or, wash stained sample and reacquire. Cannot resolve dim positive
staining from background signal.
Dirty capillary. Perform at least one cycle of
Guava Clean.
While acquiring samples, select Clean & Rinse instead of Quick Clean, and if
necessary, run it frequently.
Poor resolution between positive populations in plots displaying GRN, YLW, RED, NIR, RED2, and/or NIR2.
1. Incomplete staining with reagent(s).
2. Too much reagent in staining tube.
3. Fluorescence background too high.
4. Gains too high causing signal to bleed into other parameters.
5. Gain too low to optimally detect positive signal. 6. Background noise too high.
1. Check expiration date and amount of reagent(s) used in staining.
2. Washing cells may remove residual reagent.
3. Washing cells may remove residual reagent.
4. Adjust settings to reduce gain. Adjust compensation settings.
5. Adjust settings to increase gain. Adjust compensation settings.
6. Adjust settings to increase FSC threshold to remove debris. Or, select one of the fluorescence
parameters as the threshold.
CHAPTER 7
guava InCyte Assay
7.1 Introduction
guava InCyte was developed to be an open assay module providing all the basic tools for sample acquisition and data analysis. The guava InCyte module allows you to acquire and analyze up to six fluorescence parameters in combination with forward scatter (FSC) and side scatter (SSC), as well as area, width, and time. Unlike other flow cytometry-based software, InCyte allows you to focus analysis at either the sample or the experiment level. An experiment can be described as a single acquisition of samples, acquisition across multiple days, or multiple conditions acquired within one day.
Experimental analysis within InCyte allows you to perform both simple data set analyses, as well as more complex analyses such as; comparative analyses both within data sets (between samples) and between data sets (control vs stimulated, healthy vs disease, replicates - day 1 vs 2 vs 3) or different parameters (expression of multiple proteins or functional assays such as apoptosis).
InCyte offers the following features:
• simple gating and display of samples, including drag and drop gating capabilities • easy export of statistics
• the simultaneous application of Analysis Methods (analysis/gating strategies) across entire data sets or subsets of a data set
• visualization of statistical results in the form of a plate map
• analysis of FCS 3.0 data files acquired using any guava software module
• “grouping” which allows you to analyze subsets of samples from guava FCS files, as well as combine samples from different guava FCS files
• sample pooling for rare event analysis • post-acquisition compensation
• semi-automated compensation • IC-50 and EC-50 curves
A single Method (or analysis strategy) can be applied to multiple data sets in parallel, or multiple Methods can be applied to a single data set to obtain statistics to be displayed in a the heat map. The process of creating equation-based gating schemes has been simplified through use of drag-and-drop regions; “draggable” features are used throughout InCyte.
The HeatMap allows you to visually compare results using a plate map that displays varying shades of blue to represent relative statistical values. Results from up to six parameters or six data sets can be displayed simultaneously.
The instrument is configured to detect the following fluorochromes:
• The Green parameter has been optimally configured for FITC, GFP, or Alexa Fluor® 488.
• The Yellow parameter has been optimally configured for phycoerythrin (PE)-based, Cy3, Alexa Fluor® 532, propidium iodide (PI), TRITC, and DS Red reagents.
• The Red parameter can be used to detect PE-Cy5, PerCP, 7-AAD, PE-Cy5.5, and PI. • The NIR parameter can be used to detect PE-Cy7.
• The Red2 parameter can be used to detect Allophycocyanin (APC), Cy5, or Alexa Fluor® 647.
• The NIR2 parameter can be used to detect APC-Cy7, or APC-Alexa Fluor® 750. Additionally, all channels can be used with reagents that fluoresce in appropriate channels. Refer to "Specifications" for filter information.