TITLE: FCMPASS - Acquisition and gating of fluorescence reference materials
AUTHORS: Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [MESF Bead Calibration]
Vortex each fluorescence reference bead bottle before use.
?. [MESF Bead Calibration]
Add 1 drop (~50 µL) of each bead population to separate FACS tubes containing 250 µL of DPBS.
?. [MESF Bead Calibration]
Due to the high autofluorescence of the ‘Blank’ beads, their use is not recommended to use as 0 MESF.
?. [MESF Bead Calibration]
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.
?. [MESF Bead Calibration]
Ensure cytometer fluorescence settings are those used for small particle analysis.
?. [MESF Bead Calibration]
If the beads are >1 µm in diameter the use of a forward-scatter trigger threshold will likely yield optimal detection and reduced background.
?. [MESF Bead Calibration]
Analyze each bead sample at the same acquisition settings until >5000 bead events are recorded.
?. [MESF Bead Calibration]
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.
?. [MESF Bead Calibration]
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]
Vortex the rainbow multi-peak reference bead and MESF bead bottles before use.
?. [Multipeak Rainbow Bead Cross-Calibration]
Add 1 drop (~50 µL) of each MESF bead population to separate FACS tubes containing 250 µL of DPBS.
?. [Multipeak Rainbow Bead Cross-Calibration]
Add 1 drop (~50 µL) of the 8-peak bead population to separate FACS tubes containing 250 µL of DPBS.
?. [Multipeak Rainbow Bead Cross-Calibration]
Ensure cytometer fluorescence settings are those used for small particle analysis
?. [Multipeak Rainbow Bead Cross-Calibration]
If the beads are >1 µm in diameter the use of a forward-scatter trigger threshold will likely yield optimal detection and reduced background.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.
?. [Multipeak Rainbow Bead Cross-Calibration]
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.