Nov 09, 2022
Version 2
Flow Cytometer Fluorescence Voltration for FCMPASS V.2
- 1Translataional Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Protocol Citation: Joshua A Welsh, Sean M Cook, Jennifer Jones 2022. Flow Cytometer Fluorescence Voltration for FCMPASS. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnz4bqg5d/v2Version created by Joshua A Welsh
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2022
Last Modified: June 29, 2023
Protocol Integer ID: 72506
Abstract
Protocol to perform flow cytometer voltration to identify optimal detector settings for small particle analysis. Data acquired from this protocol are compatible with semi-automated analysis tools built into FCMPASS software.
Protocol materials
5 mL Round-bottom tubeCorningCatalog #352052
In 2 steps
8-peak rainbow beadsSpheroTech
In 2 steps
DPBSThermo Fisher ScientificCatalog #14190144
Step 2
Sample preparation
Sample preparation
10s
10s
Vortex 8-peak rainbow beadsSpheroTech bottle on a high setting for 00:00:05 .
5s
Pipette 500 µL of DPBSThermo Fisher ScientificCatalog #14190144 to two 5 mL Round-bottom tubeCorningCatalog #352052 . Label one tube 'DPBS" and the second tube 'Beads'.
Note
An observation from our protocol development is that it is important not to use a low protein binding tube for this step, as it can result in excess unbound fluor from the beads creating background noise increases leading to excessive event rate.
Add 3 drops of 8-peak rainbow beadsSpheroTech to the 'Beads' 5 mL Round-bottom tubeCorningCatalog #352052 and vortex for r 00:00:05 .
5s
Cytometer Setup
Cytometer Setup
Ensure cytometer is clean and that -Height and -Area statistics are set to be collected on all parameters and that all parameters are on.
Create a pseudocolor plot with FSC-A on the X-Axis and (488 nm) B-SSC-A on the Y-Axis and make sure both parameters are being plotted on a linear-scale.
Create a histogram plot with (405 nm) V-SSC-H on the X-Axis and make sure it is plotted on a log-scale.
Set the cytometer triggering threshold to (405 nm) V-SSC-H. All samples should be acquired with the lowest flow rate, typically ~10-15 µL min-1.
Note
Cytometer Voltage/Gain and threshold settings are subjective due to their dependency on alignment, and the scatter filters in place, amongst other variables. The following are guide values to start with and may need adjustment for optimal acquisition.
Beckman Coulter, CytoFLEX [405 nm OD0 filter, 488 nm OD2 filter]
- Threshold V-SSC-H = 1000;
- V-SSC Gain = 200
- FSC Gain = 100
- B-SSC Gain = 100
Cytek Bioscience, Aurora [405 nm OD0 filter, 488 nm OD2 filter]
- Threshold V-SSC-H = 1000;
- V-SSC Gain = 600
- FSC Gain = 70
- B-SSC Gain = 70
Acquire the DPBS tube while viewing the histogram plot from go to step #6 . Adjust the detector gain or trigger threshold until the instrument noise is being acquired at ~1000 events/sec. The instrument noise floor is distinct from detected background events in sheath as it has a sharp increase. In a system with debris there may be a tail that elongates out of this this sharp peak.
Example of threshold on the instrument noise floor
Recording this noise is not necessary as this step is identifying optimal settings.
Acquire the 'Beads' tube from go to step #3 . Using the plot from go to step #5 adjust the FSC and B-SSC gain until the single bead population is clearly visible and can be easily gated from the doublet population to the top right of it. Use the Violet SSC trigger settings identified in go to step #8
Example of clearly resolved singlet bead population
Creating a gate around the single bead population named 'Gate 1';
Adjust the stopping criteria of the instrument to record until 10,000 events are acquired on 'Gate 1' drawn in go to step #10 .
Performing Voltration
Performing Voltration
Voltration can now be performed by recording the 'Beads' tube at multiple fluorescent detector gains, leaving the trigger threshold and light scatter gains consistent. It is recommended that a recording of at least 10 fluorescent detector settings is taken. Including more increments within a voltration will result in being more confident of the subsequent optimal detector settings.
Acquisition Template.xlsx
Acquisition Template.xlsx Note
For flow cytometers with avalanche photodiodes its is recommended that the fluorescent detector settings have more incrementation at lower gains than higher gains while for instruments with photomultiplier tubes they should be spaced evenly. See template for example of settings for each tube analyses
Example Gain Voltration for CytoFLEX & Aurora
- 100
- 200
- 300
- 400
- 500
- 750
- 1000
- 1250
- 1500
- 2000
- 2500
- 3000
To ensure accurate data analysis, the brightest bead must be visible on at least two of the selected gains.