ab245835 Setup Kit V2 FirePlex® Cytometer - Abcam

Copyright © 2019 Abcam. All rights reserved

ab245835FirePlex® Cytometer Setup Kit V2

Protocol Booklet Version 1.2 June 2019For cytometer performance optimization for use with FirePlex particles.

This product is for research use only and is not intended for diagnostic use.

Version 1.2 updates:Adjustment to supplied volumes of Run Buffers I-III, in Section 3.

Copyright © 2019 Abcam. All rights reserved

Table of Contents

1. Overview 1

2. Cytometer requirements 1

3. Kit Components 2

4. Storage and Stability 2

5. Limitations 2

6. Particle Preparation for Acquisition 3

7. Post-Acquisition Analysis in Firefly Analysis Workbench (FAW)5

8. Notes 13

Technical Support 14

ab245853 FirePlex Cytometer Setup Particles Kit 1

1. Overview

This protocol contains instructions for setting up a cytometer acquire FirePlex® multiplex particles. Some cytometers may not be able to resolve the particles to a sufficient level for analysis. The Cytometer Setup Kit is designed to help identify properly resolving machines for FirePlex® multiplex assays, as well as the appropriate Run Buffer to be used on a given instrument. If you have questions about compatibility, contact our Technical Support team at [email protected].

2. Cytometer requirements

Digital cytometer with one of the either configuration:

o 488 nm (Blue Laser) excitation for “Green”, “Yellow”, “Red” fluorescence [recommended]

Green Filter (e.g. FITC, 530/30 Bandpass filter). Yellow Filter (e.g. PE, 575/25 Bandpass filter). Red Filter (e.g. PE-Cy5, 685/35 Bandpass filter; or

PerCP-Cy5.5, 695/40 Bandpass Filter).

Fluorochrome PMT Channel Excitation Emission

FITC - Green FL1 488 nm 530/30 nm PE - Yellow FL2 488 nm 575/25 nm

PerCP-Cy5.5 - Red FL3 488 nm 695/40 nm

OR

488 nm (Blue Laser) excitation for “Green” and “Red” fluorescence; and 561 nm (Yellow/Green Laser) excitation for “Yellow” fluorescence.

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ab245853 FirePlex Cytometer Setup Particles Kit2

3. Kit Components

Please read these instructions carefully prior to beginning the assay.

The Cytometer Setup Kit is supplied with all reagents required to assess the compatibility of a given flow cytometer, with the FirePlex assays. This kit also enables users to identify the appropriate Run Buffer to be used for their particular flow cytometer.

Cytometer Setup Particles, 700 µL. 96-well Filter Plate Run Buffer I, 1.5 mL Run Buffer II, 2 mL Run Buffer III, 2 mL 1x PBS, 2 mL

Note: After running the Cytometer Setup Kit, users should note the Run Buffer compatible with their flow cytometer. Run Buffers I-III and PBS are not supplied with FirePlex miRNA assay or FirePlex immunoassay products and must be purchased separately using the indicated product codes below.

Item Quantity

Run Buffer I (ab245836) 1x96 tests

Run Buffer II (ab234450) 1x96 tests

Run Buffer III (ab245837) 1x96 tests

PBS (no calcium, no magnesium, user supplied, e.g. Gibco #1419250 or other

similar grade solution)—


4. Storage and Stability

Store kit at 4°C in the dark immediately upon receipt. Kit has a storage time of 6 months from receipt.

5. Limitations

Assay kit intended for research use only. Not for use in diagnostic procedures.

— PROTOCOL CONTINUES ON THE NEXT PAGE —


6. Particle Preparation for Acquisition

6.1 Locate the 4 bottles of reagents provided with the kit. Reagent bottles are acquisition buffers called Run Buffer I, Run Buffer II, Run Buffer III and 1x PBS.

6.2 Apply 170 µL of the 4 acquisition buffers to 10 wells of the provided 96-well filter plate using the suggested plate layout below. Note that 4 wells will be resuspended in 1x PBS (A1-B2), and 2 wells each for Run Buffers I (C1-C2), II (D1-D2) and III (E1-E2).


6.3 Vortex the container of Cytometer Setup Particles for 5 seconds and invert several times.

6.4 To each well containing 170 µL of acquisition buffer, add 35 µL of Cytometer Setup Particles. Each well will now have 205 µL total volume.

6.5 Mix the plate by placing on an orbital shaker with 2-3 mm orbital diameter for 2 minutes at 750 RPM, or mix by pipetting each well with 100 µL of aspiration volume. If mixing by pipetting, try not to introduce large bubbles of solution within the wells.

6.6 Using one of the PBS resuspension wells first (e.g. A1 or A2), acquire on your cytometer using “Green” fluorescence (e.g. FITC, or FL1) as the “Threshold” / “Trigger” parameter (as opposed to the standard “FSC” parameter that is normally used for the Threshold/Trigger).


6.7 Adjust voltages on your cytometer using one or two of the PBS resuspension wells such that you can resolve the particles similar to the example below from a BD LSRII machine.

6.8 Once you have general voltages on scale for your machine and are seeing the expected 3 peaks on the Green Channel and have your Yellow and Red channels below saturation point, proceed to doing an automated acquisition of the 8 remaining wells of resuspended setup particles (2 each of PBS and Run Buffers I, II, III).

6.9 Export all wells as FCS 3.0 files for subsequent import into the FirePlex Analysis Workbench for file decoding and instrument sensitivity assessment.

NOTE: Please consult our cytometer-specific protocols for more detailed setup instructions:http://www.abcam.com/protocols/flow-cytometry-protocols-for-multiplex-mirna-assays

http://www.abcam.com/protocols/flow-cytometry-protocols-for-multiplex-mirna-assays

http://www.abcam.com/protocols/flow-cytometry-protocols-for-multiplex-mirna-assays


7. Post-Acquisition Analysis in FirePlex Analysis Workbench (FAW)

7.1 Download the FirePlex™ Analysis Workbench (FAW) onto your personal computer from http://www.abcam.com/kits/firefly-analysis-workbench-software-for-multiplex-mirna-assays.

7.2 Open the FAW program. Click the “Load FCS” icon, or use the File Menu to select “Load FCS, FCI, or FWS”.

7.3 With the first time launching, you will be prompted with the “Assay Mode” window. Select the “immune” for Assay mode menu and “expert” for Features shown menu. Optional to select the “Remember my choice” button. Click OK.

7.4 You will be prompted to locate the FCS files you wish to import (at least one file from each suspension buffer condition). Once located on your computer or external drive, select all the files you wish to import and click “Open”.

http://www.abcam.com/kits/firefly-analysis-workbench-software-for-multiplex-mirna-assays

http://www.abcam.com/kits/firefly-analysis-workbench-software-for-multiplex-mirna-assays


7.5 You will be prompted with the following “Probe Definition: Panel Barcode” screen. Click the “Select PLX built-ins & history” button.

7.6 A “Panel Selection” window will open. Locate the line named “[24 probes] ab245835 – Cytometer Setup Kit V2” and click the “OK”.

7.7 The FCS files will load and the Upper Left section of screen will display the FCS file name and an assigned “QC by Count” pie-chart score will display in the wells.


7.8 Locate the “Analyze” menu and select the “Modify Settings” option.

7.9 A “FCS Decoding Settings” menu will appear.


7.10 Check that the correct PMT descriptions from your cytometer are being assigned to the correct “green”, “yellow”, and “red” Channel names in this window. If the automatic assignments are incorrect, you can select the correct channel using the drop-down selection option in each color channel selection. Click “OK” when complete.

7.11 Double-click on any of the wells in the plate view. An “Inspect and change parameters” window will appear. Locate the “Code QC Plot” and “QC Table”


7.12 Click on the “QC Table” tab to bring up the decoding statistics information. Locate the “total yield” column, and then identify the wells that have the highest percentage value. This line should also correspond to the highest value in the “# p decoded” column. The Run Buffer (I, II, III or PBS) that has the highest Total Yield will in this QC Table should yield the most efficient decoding of the FirePlex particles on your particular cytometer.

7.13 Make a note of which of the Run Buffers results in the highest Total Yield for your cytometer. Click OK on the “Inspect and change parameters” window to exit.

7.14 Left click on all the wells to highlight them light-blue. Click on the dashed box icon to “Make experiment from selected samples”. The red reporter levels will now display in the Upper Right section of the screen (in MFI) for each of the 24 particle positions (A). The graphs will initially be in Linear format. 6 bars each have a High, Medium, Low or Null expression labels. Click the “Log Scale” display option directly above the graph to change the MFI scale to display in Log10 format (B).

A.


B.

7.15 Locate the “ymin” and “ymax” boxes above the bar graphs. You can enter in specific MFI values here to fix the Y-axis scale display (e.g. MFI values of ymin = 10,000 and ymax = 100,000). You should be able to distinctly visual the 4 different expression level groups on the modified log scale.

Note: If you do not see all 24 groups present with similar MFI expression levels in High, Medium, Low and Null groups, contact our Technical Support team at [email protected].



7.16 After completing the visual check for correct population and expression level resolution, you will export the data for linearity calculation. Locate the Export icon that displays “Save workspace or export all experiments to spreadsheet”. Click the icon and designate a name and location for the file to be saved to.

7.17 An “Export Options Form” window will appear. Keep the 3 selected options checked in the 2 tabs.


7.18 Open the CSV file using Microsoft Excel. The MFI values of the High, Medium, Low and Null groups for the different wells (buffer conditions) will appear under their respective code names (e.g. High-1, Medium-2, etc.).

7.19 Using Excel, calculate the Average MFI Values of the High, Medium, Low, and Null groups. The corresponding concentrations of reporter fluorophore for these positively-labeled groups are 1600, 80 and 4 ng/ml respectively.

7.20 Check to make sure that the ratio of average MFI of the Low and Null groups is greater than 2.5 (MFILow:MFINull > 2.5). If your MFILow:MFINull ratio is less than 2.5, try reacquiring the Cytometer Setup Particles again with higher Red channel voltage on your cytometer and perform Steps 6.1-6.9 again.


7.21 Calculate the Log transformations of the MFI and the Concentration values.

7.22 Create a scatter plot of the Log transformed data. Add a Trendline to the plot and have it display the equation on the chart and the R2 value.

7.23 Both the Slope of the Trendline and the R2 value should equal about 1. If your Slope and R2 values significantly depart from equaling 1 (e.g. >20% deviation for either), please contact our Technical Support team at [email protected].



8. Notes

ab245853 FirePlex Cytometer Setup Particles Kit 18

Technical Support

Copyright © 2019 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.

FirePlex® is a registered trademark in the United States and is an unregistered trademark elsewhere.

For technical support inquiries, please contact [email protected]


ab245835 Setup Kit V2 FirePlex® Cytometer - Abcam

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