3. RECOLECCIÓN DE DATOS
3.1 Precauciones importantes
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.
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 ExpressPlus Assay Troubleshooting
DetectionDisplays the laser status, the SSC (if installed) GRN, YLW, and RED voltage settings, and the FSC gain.
NOTE: Do not change the voltages from this panel. Use the
sliders in Adjust Settings to adjust the SSC, GRN, YLW, and RED voltages.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 ExpressPlus 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 ExpressPlus FCS file to another directory or give it a new name. guava ExpressPlus
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, or RED.
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, and/ or RED.
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 Plot 1 but
not in Plot 2 or Plot 3.
Plot 1 gate excludes events, and gate is applied to Plot 2 and/or Plot 3.
Uncheck Plot 1 (under Gated On) for Plots 2 and 3 to see if events appear. Or, set Plot 1 gate to include population of interest.
Events appear in Plot 2 but not in Plot 3.
Plot 2 gate excludes events, and gate is applied to Plot 3.
Uncheck Plot 2 (under Gated On) for Plot 3 to see if events appear. Or, set Plot 2 gate to include population of interest. Events appear off scale in
dot plots or histograms.
FSC gain or SSC, GRN, YLW, and/or RED voltages set incorrectly, or samples staining brightly.
Adjust gain setting or voltage settings so positive
populations appear on scale. Repeat Adjust Settings with negative sample. Adjust compensation settings. Poor resolution between
positive and negative populations.
1. Voltages 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.
Poor resolution between positive populations in plots displaying GRN, YLW, and/ or RED.
1. Incomplete staining with reagent(s).
2. Too much reagent in staining tube.
3. Fluorescence background too high.
4. Voltage too high causing signal to bleed into other parameters.
5. Voltage 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 voltage. Adjust
compensation settings. 5. Adjust settings to increase
voltage. Adjust
compensation settings. 6. Adjust settings to increase
FSC threshold to remove debris. Or, select one of the fluorescence
parameters as the threshold.
CHAPTER 6
guava ExpressPro Assay
Introduction
The guava ExpressPro Assay allows you to acquire and analyze up to six fluorescence parameters in combination with forward scatter (FSC) and side scatter (SSC), provided as an optional parameter, as well as area, width, and time, to identify cells with specific phenotypic markers or specific properties. The software provides absolute cell counts and allows you the flexibility to stain samples with the guava Express Reagents, your own fluorochrome-conjugated antibody reagents, DNA intercalating dyes, or other green, yellow, red, and/or near infrared (NIR) fluorescent reagents that are excited by a 488-nm laser, as well as red (RED2) or near infrared (NIR2) fluorescent reagents that are excited by a 640-nm laser.
The guava ExpressPro Assay can be used for any one-, two-, three-, four-, five-, or six- color assay including:
• protein expression or other cell-surface marker experiments • intracellular staining
• screening and analyzing cells expressing Green Fluorescent Protein (GFP) The Green parameter can be used for FITC, GFP, or Alexa Fluor 488. The Yellow parameter has been optimally configured for use for phycoerythrin (PE)-based reagents, although fluorochromes with comparable emission spectra, such as propidium iodide (PI), TRITC, DS Red, sulforhodamine, Cy3, and Alexa Fluor 532 can also be used. 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 or PE-AlexaFluor 750. The RED2 parameter can be used to detect Allophycocyanin (APC) or fluorochromes with comparable emission spectrum. The NIR2 parameter can be used to detect Cyanine 5 (Cy5), APC-Cy7, APC- AlexaFluor 750, or fluorochromes with comparable emission spectrum. Refer to
Specifications for the guava easyCyte HT System fluorescence detection range. In addition to the height value for each pulse measured, you can select to save width and area of each pulse for a single parameter. These measurements are especially useful in cell cycle analysis to discriminate doublets from singlets.
To run the assay, stain samples using an appropriate fluorochrome combination for up to six fluorescence parameters. Acquire the samples on the guava easyCyte HT System using guavaSoft Software. The data are displayed in eight plots, which can be set up to show histograms or dot plots with any parameter combination (FSC, SSC, GRN, YLW, RED, near infrared [NIR], RED2, and NIR2, as well as area, width, and time). You may set up to 16 global regions and 32 global gates to select a specific subpopulation(s) for
analysis, then choose the gate(s) for which you want to display statistics for the remaining plots.
Histogram and dot plot statistics include the cell count, cells/mL, mean signal intensity, geometric mean signal intensity, median signal intensity, and %CV for the data within the entire plot and the subset of data within the markers/gate. Additionally, the data are expressed as a percentage of the total data within the plot (whether gated from the dot plot or histogram) and as a percentage of data within the gate.
The guava ExpressPro data for all samples within a data set are saved to an FCS 3.0 file. The data can be analyzed immediately after the sample is acquired using guavaSoft Software, or later using guavaSoft Software or an FCS 2.0–compatible program, if you selected to save FCS 2.0 files. In addition to the saved data file, user-selectable statistics, instrument settings, and the acquisition summary information are exported to a
spreadsheet file.