ab Firefly Cytometer Setup Particles

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1 Version 1 Last updated 12 October 2016 ab Firefly 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

2 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 Copyright 2016 Abcam. All rights reserved

3 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 technical@abcam.com. 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.

4 3. Kit Components Please read these instructions carefully prior to beginning the assay. Cytometer Setup Particles, 360 µl. Microdilution Tubes (1.2 ml, polypropylene), 10 units 1X PBS, 4 ml 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. 2

5 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). 3

6 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: 4

7 7. Post-Acquisition Analysis in Firefly Analysis Workbench (FAW) 7.1 Download the Firefly Analysis Workbench (FAW) onto your personal computer from 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. 5

8 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. 6

9 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 dropdown selection option in each color channel selection. Click OK when complete. 7

10 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. 8

11 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 technical@abcam.com 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. 9

12 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 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 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. 10

13 7.15 Check to make sure that the ratio of average MFI of the Low and Null groups is greater than 2.5 (MFI Low :MFI Null > 2.5). If your MFI Low :MFI Null 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 Calculate the Log transformations of the MFI and the Concentration values 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 R 2 value. 11

14 7.18 Both the Slope of the Trendline and the R 2 value should equal about 1. If your Slope and R 2 values significantly depart from equaling 1 (e.g. >10% deviation for either), please contact our Technical Support team at technical@abcam.com. 12

15 8. Notes 13

16 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. Austria wissenschaftlicherdienst@abcam.com France supportscientifique@abcam.com Germany wissenschaftlicherdienst@abcam.com Spain soportecientifico@abcam.com Switzerland technical@abcam.com Deutsch: Français: UK, EU and ROW technical@abcam.com +44(0) Canada ca.technical@abcam.com US and Latin America us.technical@abcam.com Asia Pacific hk.technical@abcam.com (852) China cn.technical@abcam.com Japan technical@abcam.co.jp +81-(0) Singapore sg.technical@abcam.com Australia au.technical@abcam.com +61-(0) New Zealand nz.technical@abc.com +64-(0)

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