ab211043 Setup Particles Firefly® Cytometer · 2017. 4. 28. · ab211043 Firefly Cytometer Setup...

Copyright © 2016 Abcam. All rights reserved

Version 1 Last updated 28 April 2017

ab211043Firefly® Cytometer Setup Particles

For cytometer performance optimization for use with Firefly Multiplex particles.

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

Copyright © 2016 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

ab211043 Firefly Cytometer Setup Particles Kit 1

1. Overview

This protocol contains instructions for setting up a cytometer acquire Firefly® multiplex particles. Some cytometers may not be able to resolve the particles to a sufficient level for analysis. The Cytometer Setup Particles are designed to help identify properly resolving machines for Firefly® multiplex assays. 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).

OR

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

mailto:[email protected]

ab211043 Firefly Cytometer Setup Particles Kit2

3. Kit Components

Please read these instructions carefully prior to beginning the assay.

Item Quantity Storage Condition

Cytometer Setup Particles 360 µL +4ºC

Microdilution Tubes (1.2 mL, polypropylene) 10 units +4ºC

1X PBS 4 mL +4ºC

Run Buffer 4 mL +4ºC

Tungsten Wire (9” Length) 1 +4ºC

4. Storage and Stability

Store kit at 4°C in the dark immediately upon receipt. Kit has a storage time of 1 year from receipt.

5. Limitations

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


6. Particle Preparation for Acquisition

6.1 Pipet 200 µL of 1X PBS into a microdilution tube (provided), or pipet 170 µL into a well if use a 96-well plate (not provided, for use in plate-loading cytometers).

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

6.3 Pipet 35 µL mixed Cytometer Setup Particles into the microdilution tube (or well) prepared in step 1. Mix by pipetting.

6.4 Just prior to loading the sample on your cytometer, pulse vortex the microdilution tube (capping the open top with your gloved finger) and gently drop the microdilution tube into a 5 mL polystyrene tube (not provided). Load the sample onto the Sample Injection Port (SIP) of the cytometer.

6.5 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).


6.6 Adjust voltages on your cytometer such that you can resolve the particles similar to the example below.

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


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

7.1 Download the Firefly™ 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. Once launched, load the FCS file you generated in Step 6 of this protocol by using the icon below, or clicking “File” menu, then selecting “Open FCS”.

7.3 Select your FCS file for opening. You will be prompted with the following “Probe Definition: Panel Barcode” screen. For first time use, select the “Browse for PLX file” button.

7.4 Locate the PLX file that is associated with your particle mix’s lot number. Select the file for opening. Once loaded, click the “OK” button on the “Probe Definition: Panel Barcode” screen.

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


7.5 The FCS will load and the Upper Left section of screen will display the FCS file name and an assigned “QC” score (A). Left click the file name to highlight it Blue (B). Left click on the dashed box icon to “Make experiment from selected samples” (C).

A. B.

C.

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


7.7 A “FCS Decoding Settings” menu will appear.

7.8 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.


7.9 The Upper Right section of the screen will now display the fluorescent reporter levels (in MFI) for each of the 24 particle positions (A). The graphs will initially be in Linear format. 5 bars each have a High, Medium, or Low expression label; 9 bars have a Null expression label. Click the “Log Scale” display option directly above the graph to change the MFI scale to display in Log10 format (B).

A.

B.


7.10 Using your computer mouse’s or trackpad’s scroll wheel function hovering over the MFI scale on the left side of graph, you can adjust the MFI scale display so that the Null samples are just above the axis and the High samples are just below the top of the scale display. You should be able to distinctly visual the 4 different expression level groups on the modified log scale.

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].

7.11 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 CSV file(s)”. Click the icon and designate a name and location for the CSV file to be saved to.



7.12 You will be prompted with an “Export Options Form” window. Under the “Options” tab, select only “Write all experiments to one file” (A). Under the “Statistics” tab select only “Export Signal” (B).

A. B.

7.13 Open the CSV file using Microsoft Excel. The MFI values of the High, Medium, Low and Null groups will appear in row 3 under their respective populations.

7.14 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.


7.15 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 8-17 again.

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

7.17 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.


7.18 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. >10% deviation for either), please contact our Technical Support team at [email protected].



8. Notes

ab211043 Firefly Cytometer Setup Particles Kit 14

Technical Support

Copyright © 2016 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.

[email protected] | [email protected] | [email protected] | [email protected] | 91-114-65-60

[email protected] Deutsch: 043-501-64-24 | Français: 061-500-05-30UK, EU and [email protected] | +44(0)1223-696000

[email protected] | 877-749-8807US and Latin [email protected] | 888-772-2226

Asia Pacific [email protected] | (852) [email protected] | +86-21-5110-5938 | [email protected] | +81-(0)3-6231-0940Singapore [email protected] | 800 188-5244

[email protected] | +61-(0)3-8652-1450New Zealand [email protected] | +64-(0)9-909-7829

ab211043 Setup Particles Firefly® Cytometer · 2017. 4. 28. · ab211043 Firefly Cytometer Setup...

Documents

Transcript of ab211043 Setup Particles Firefly® Cytometer · 2017. 4. 28. · ab211043 Firefly Cytometer Setup...