Aug 05, 2020
FCMPASS - Acquisition and gating of fluorescence reference materials
- 1Translataional Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health
- Translational Nanobiology Section
Protocol Citation: Joshua A Welsh, Jennifer Jones 2020. FCMPASS - Acquisition and gating of fluorescence reference materials. protocols.io https://dx.doi.org/10.17504/protocols.io.bjcpkivn
Manuscript citation:
Welsh J A, Jones J C,Small Particle Fluorescence and Light Scatter Calibration Using FCMPASSSoftware,Current Protocols in Cytometry, 94, e79. doi: 10.1002/cpcy.79
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: August 05, 2020
Last Modified: June 29, 2023
Protocol Integer ID: 40047
Keywords: flow cytometry, extracellular vesicles, calibration,
Disclaimer
This protocol summarizes key steps for a specific type of assay, which is one of a collection of assays used for EV analysis in the NCI Translational Nanobiology Section at the time of submission of this protocol. Appropriate use of this protocol requires careful, cohesive integration with other methods for EV production, isolation, and characterization. By using the FCMPASS software you agree to the following terms and conditions.
Terms & Conditions of use for FCMPASS software.
Definitions: The term “SOFTWARE” throughout this agreement means the machine readable, binary, object code form, and the related documentation for FCMPASS, a software package that is designed to allow flow cytometer calibration for small particles. The term “RECIPIENT” means the party that downloads the software. The term “PROVIDER” means the National Cancer Institute (NCI), a participating institute of the National Institutes of Health (NIH), and an agency of the United States Government.By downloading or otherwise receiving the SOFTWARE, RECIPIENT may use the SOFTWARE subject to RECIPIENT’s agreement to the following terms:
- THE SOFTWARE SHALL NOT BE USED IN THE TREATMENT OR DIAGNOSIS OF HUMAN SUBJECTS. RECIPIENT is responsible for compliance with all laws and regulations applicable to the use of the SOFTWARE.
- RECIPIENT shall not distribute the SOFTWARE, in whole or in part without express advance written approval of PROVIDER.
- The SOFTWARE may be used for research, academic, and educational purposes only. The SOFTWARE may not be used for commercial purposes. RECIPIENT will not license or sell or use the SOFTWARE for commercial purposes or applications.
- The SOFTWARE that is distributed pursuant to this Agreement has been created by United States Government employees. In accordance with Title 17 of the United States Code, section 105, the SOFTWARE isnot subject to copyright protection in the United States. Other than copyright, all rights, title and interest in the SOFTWARE shall remain with the PROVIDER.
- RECIPIENT shall not modify, extend, decompile, make derivatives of, merge, publish, reverse engineer or distribute the SOFTWAREwithout written permission from PROVIDER.
- RECIPIENT may publish or otherwise publicly disclose the results of using the SOFTWARE. RECIPIENT agrees to acknowledge PROVIDER’s contribution of the SOFTWARE in all written publications containing any data or information regarding or resulting from use of the SOFTWARE.
- THE SOFTWARE IS PROVIDED "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT ARE DISCLAIMED. IN NO EVENT SHALL THE PROVIDER OR THE INDIVIDUAL DEVELOPERS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE. PROVIDER makes no representations that the use of SOFTWARE will not infringe any patent or proprietary rights of third parties.
- RECIPIENT may, on an as-needed basis, send to PROVIDER reports regarding the application of the SOFTWARE and the effectiveness and problems encountered in using the SOFTWARE, without disclosing RECIPIENT’s confidential information. Information from general reports may be used by the PROVIDER to enhance the capabilities of the SOFTWARE. Reports can be forwarded to the PROVIDER at one of the following addresses: joshua.welsh@nih.gov or jennifer.jones2@nih.gov
- No indemnification for any loss, claim, damage, or liability is intended or provided by either Party under this Agreement. Each Party shall be liable for any loss, claim, damage, or liability that said Party incurs as a result of said Party's activities under this Agreement, except that Provider, as an agency of the United States, assumes liability only to the extent as provided under the Federal Tort Claims Act (28 U.S.C. Chapter 171 Sections 2671-2680).
- RECIPIENT agrees not to claim, infer, or imply endorsement by the United States Government, or any of its organizational units, contractors or employees. RECIPIENT agrees not to use any trademarks, service marks, trade names, logos or product names of NCI or NIH to endorse or promote products derived from the SOFTWARE without specific, prior and written permission.
- Title in the SOFTWARE shall remain with the PROVIDER. It is understood that nothing herein will be deemed to constitute, by implication or otherwise, the grant to either party by the other of any license or other rights under any patent, patent application or other intellectual property right or interest. PROVIDER reserves the right to distribute the SOFTWARE to others and to use it for PROVIDER’s own purposes. The United States Government explicitly retains all rights to use the SOFTWARE for any purpose, to have it used on the Government’s behalf or to allow others to use it.
Abstract
This protocol outlines the steps required to acquire fluorescence reference material data for use with the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.
Materials
MATERIALS
Flow Cytometer
Vortex
Rainbow calibration beadsSpheroTechCatalog #RCP-30-5A
DPBSInvitrogen - Thermo FisherCatalog #14190
Falcon® 5 mL Round Bottom Polystyrene Test Tube with Snap Cap Sterile Individually Wrapped CorningCatalog #352003
Fluorescence Calibration Reference Material
MESF Bead Calibration
MESF Bead Calibration
Vortex each fluorescence reference bead bottle before use.
Add 1 drop (~50 µL) of each bead population to separate FACS tubes containing 250 µL of DPBS.
Due to the high autofluorescence of the ‘Blank’ beads, their use is not recommended to use as 0 MESF.
Many commercially available fluorescence calibration beads are bright and will require extrapolation instead of interpolation to obtain the dim fluorescence values. The accuracy of the extrapolation will therefore be influenced by a number of factors including the gating of the populations. While less ergonomic, it is preferable to analyze 1 bead population at a time. This allows for gating on scatter parameters, rather than fluorescence parameters, making the statistics less biased by the gating strategy. Analyzing one bead population at a time will also minimize the subjectivity when gating fluorescence populations that overlap, sometimes causing small peaks.
Gating fluorescence reference beads.A) Gating of bead population using FSC-A and SSC-A. B) Histogram of all four APC MESF bead population in a cumulative distribution. Arrows highlight areas of overlap between beads that may lead to subjectivity on where to manually draw gates. C) Histogram of individual APC MESF bead populations.
Ensure cytometer fluorescence settings are those used for small particle analysis.
If the beads are >1 µm in diameter the use of a forward-scatter trigger threshold will likely yield optimal detection and reduced background.
Analyze each bead sample at the same acquisition settings until >5000 bead events are recorded.
Gate each bead population on FSC-A vs. SSC-A and obtain the median area statistic for the fluorescence parameter being calibrated to move on to the 'FCMPASS Fluorescence Calibration' protocol.
By default, flow cytometers trigger the acquisition of an event using the pulse height parameter. In cases where a trigger threshold is being defined e.g. SSC. It is recommended that the pulse-height is used so that the limit of detection can be defined in calibrated units. There is no consensus within the small particle community over the use of pulse height vs. area. We recommend that, in general, if the parameter being calibrated was not used as a trigger channel the pulse area statistic should be used due to it tending to be linear at low signal intensities and therefore a more reliable method for extrapolation.
Multipeak Rainbow Bead Cross-Calibration
Multipeak Rainbow Bead Cross-Calibration
Vortex the rainbow multi-peak reference bead and MESF bead bottles before use.
Add 1 drop (~50 µL) of each MESF bead population to separate FACS tubes containing 250 µL of DPBS.
Add 1 drop (~50 µL) of the 8-peak bead population to separate FACS tubes containing 250 µL of DPBS.
Ensure cytometer fluorescence settings are those used for small particle analysis
If the beads are >1 µm in diameter the use of a forward-scatter trigger threshold will likely yield optimal detection and reduced background.
Analyze each bead sample at the same acquisition settings until >2000 bead events are recorded. For the 8-peak beads this will be >16,000 events.
Gate each MESF bead population on FSC-A vs. SSC-A and obtain the median statistic for the parameter and perform calibration of the MESF and 8-peak bead files.
Once the 8-peak rainbow beads are calibrated in PE MESF units, gate the population on FSC-A vs. SSC-A to obtain singlets. Using the singlet population gate each of the 8-peak populations.
The gating the individual fluorescent bead populations can be done in the parameter which best separates each population. This may be a different fluorescence detector than the calibrated parameter. While the gating of each population does not have to be on the MESF parameter itself, the MESF parameter should be checked to ensure all populations are on scale. In some 3rdparty software the scale limits (minimum and maximum value) will influence the outputted statistic due to how the data is binned.
Once each of the 8-peak populations has been gated, obtain the median MESF value for each of the populations. These values are now the cross-calibrated values for these beads and can be used on the same instrument at different gains.
Gating and cross-calibration of fluorescence reference beads.A) Gating of 8-peak bead population using FSC-A and SSC-A. B) Regression of PE MESF bead reference values vs. acquired arbitrary statistics for each population. C) Histogram of gated (Panel A) 8-peak reference beads and gating of each population (red). D) Histogram of gated (Panel A) 8-peak reference beads converted to PE MESF units using regression (Panel B). E) Histogram of gated PE MESF beads and gating of each population (red). F) Histogram of gated PE MESF beads converted to PE MESF units using regression (Panel B).
If an instrument is re-aligned or filters are changed these values will no longer be valid and will require cross-calibration to be performed again. In general, it is good practice to regularly cross-calibrate 8-peak bead reference values, e.g. once a month.