TITLE: Astrios EQ instrument setup and sample acquisition 
AUTHORS: Aizea Morales-Kastresana, Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol the authors describe the general steps to follow in order to achieve optimallaser:stream:detectors alignment and sample acquisition on the Astrios EQ flow cytometer (Beckman Coulter). The general concept however can be applied to other jet-in-air instruments.</div></div>

[STEPS]
?. Turn on the instrument at least an hour before running the samples. Let the pressure and stream to stabilize and prime the fluidics system to remove bubbles in the circuit. Turn lasers on and allow them to warm up whilst shuttered.
?. Wash the sample line with FACS Rinse solution for about 20 minutes at a high differential pressure (1 psi over the sheath pressure). Repeat same procedure with clean DPBS for 20 more minutes.
?. Adjust the vertical alignment.
Note: To test whether the stream is vertical or not, raise the nozzle while looking at the relative position of the stream to the pinholes. If the position doesn’t change, the stream is vertically well aligned; if it does change, then the verticality must be tweaked. A good vertical alignment maximizes the detection of parameters in all laser paths.
?. Set the triggering threshold to the 561-SSC channel. Adjust the triggering threshold channel and voltage to allow the visualization of the noise population, to approximately 10,000 events per second.
Note: The noise is a random representation of the diffusely scattered photons from the laser beam and stream intercept.Because of the high event rate of these low-level signals, inclusion of these events on some instruments is only feasible in a very limited way due to limitations in their baseline restoration algorithms and other signal processing attributes.Background noise is informative because: i) it serves as a window into the population of EVs that fall under the triggering-threshold; ii) it allows the determination of too much free dye in the interrogation point; and iii) it helps to identify when EV samples are being analyzed at a concentration that is too high and is therefore at risk of coincident detection(Morales-Kastresana et al., 2017; van der Pol, van Gemert, Sturk, Nieuwland, & van Leeuwen, 2012). For these reasons, the authors refer to the noise as “background reference noise”.
?. Load a sample containing a mix of 200 nm yellow and red beads at 1x107beads ml-1(about a 1x106-fold dilution of original stock) to fine-tune the stream:laser alignment.
Note: Any combination of beads that are excited by different lasers can be used. The goal is to have two populations, whose fluorescence will be collected in different pinholes, in a way that the stream is aligned according to two pinholes. This ensures the correct vertical alignment.
Note: The dimmer the fluorescent beads used, the better the alignment will be for EVs.
?. Open a dot plot depicting yellow (~515 nm) and red (~605 nm) fluorescent channels in each axis (or correspondent fluorescent axis for the chosen beads). Tweak the alignment until the fluorescence signal is optimized for both bead sets. While doing this, try to keep the total event rate, including the noise, around 10,000-20,000.
Note: Alignment will be optimal when the distance between the noise and bead populations is the biggest in terms of fluorescence, while the bead population remains as tight as possible. Also, the event rate should not increase significantly with respect to DPBS alone, since the contribution of the beads to the overall rate is insignificant.
Note: It is very useful to monitor Time versus any parameter (fluorescence and scatter) to determine how the distance between noise and beads varies with alignment adjustments.
Note: If the bead populations and the reference noise population appear as “split” populations, there is probably drop-drive noise (especially with the 70 µm nozzle), which can be eliminated by dropping the break off point by lowering the amplitude or frequency, to the extent possible while maintaining stable Intelli-Sort settings and a stable breakoff point.
?. Once aligned, acquire and save a representative sample of the beads used for the alignment.
This file will serve as a reference for future alignment, allowing comparisons between the alignment between experiments.
?. Wash the sample line with DPBS for 5 minutes at 1 psi.
Note: As the sample lines clear, monitor the disappearance of the 200 nm beads from the alignment steps over time by referring to the time versus scatter or fluorescence plots. If alignment beads do not disappear completely, increase the pressure up to a differential pressure of 1.4. If beads are still observed, there may be contaminating beads in the sample path (either in the lines, junction points between fittings, or in the nozzle reservoir), and you will need to clear these residual beads before continuing. It can be useful to wash the line with rinse solution and followed by DPBS for 5 minutes each. Once washing is done, change rinse and DPBS tubes for future uses, as these solutions may contain contaminating beads from the sample injection tube.
?. Save an acquisition of 30 seconds of clean DPBS.
?. Load the EV sample, increase pressure up to 1 psi to accelerate the sample loading and then decrease the differential pressure to 0.3 psi.
Note: A 0.3 psi differential pressure is offered here as a reference for what commonly works on AstriosEQ sorters that we have worked with. A differential pressure that permits stable detection of single EVs, avoiding coincidence, should be selected. Authors recommend keeping the differential pressure the same for all samples to be analyzed during an experiment, since event rate can be a valuable source of information.
?. Wait until the event rate is stable (use the time versus scatter plot). Then stop the acquisition in the software (not the machine) and start acquiring again for 30 seconds. Save the acquisition.
Note: Authors recommend saving acquisitions of the same duration, in order to compare event numbers among samples.
?. Wash the sample line with DPBS for 5 minutes at 1 psi.
?. Run samples, save acquisitions and wash with DPBS, as described above.Keep samples at 4°C and in the dark.
?. Wash the sample line with FACSClean, FACSRinse and DPBS consecutively, for 10 minutes each at 2 psi. Turn off the lasers and fluidics. Rinse the nozzle and place a FACS tube cap with some clean water under the nozzle tip.
?. [Troubleshooting]
ABC1ARTIFACTSOURCEWHAT TO DO2False positive signalDrop drive noiseReduce amplitude and/or frequency3Nonspecific binding of antibodiesAdd control EVs that do NOT express the target antigen&nbsp;4Antibody/dye aggregatesAdd control EVs that do NOT express the target antigen&nbsp;5Add control antibody/dye alone6Background antibody/dye fluorescenceAdd control antibody/dye alone7Noise fluorescence shifting8Low resolution of positive signal over the noise/negative populationBackground antibody/dye fluorescenceWash the excess antibody/dye9No positive signal detectionLow antigen densityUse a more sensitive instrument, brighter fluorochrome and/or brighter staining method10Low antibody affinityIncrease concentration and/or incubation time11Antibody/fluorochrome conjugate not working properlyChange antibody/clone/fluorochrome/lot12Unexpected increase in event rateChange in differential pressure of sheath tank and sample lineTime vs scatter parameter to identify these peak13Stuck material in sample line/nozzleFlush sample line with detergent and PBS with increased sample pressure differential (boost).14Contaminating material from previous sampleRun filtered PBS before and after samples, to minimize the presence of contaminating particles.15Coincident detection of particlesSample is too concentratedDilute the sample16Use spike in beads to ensure an operational concentration range17Nanobubbles at the interrogation pointAre created due to the high pressure in the nozzleRun PBS control between samples to have that source of noise identified and controlled
ABC1ARTIFACTSOURCEWHAT TO DO2False positive signalDrop drive noiseReduce amplitude and/or frequency3Nonspecific binding of antibodiesAdd control EVs that do NOT express the target antigen&nbsp;4Antibody/dye aggregatesAdd control EVs that do NOT express the target antigen&nbsp;5Add control antibody/dye alone6Background antibody/dye fluorescenceAdd control antibody/dye alone7Noise fluorescence shifting8Low resolution of positive signal over the noise/negative populationBackground antibody/dye fluorescenceWash the excess antibody/dye9No positive signal detectionLow antigen densityUse a more sensitive instrument, brighter fluorochrome and/or brighter staining method10Low antibody affinityIncrease concentration and/or incubation time11Antibody/fluorochrome conjugate not working properlyChange antibody/clone/fluorochrome/lot12Unexpected increase in event rateChange in differential pressure of sheath tank and sample lineTime vs scatter parameter to identify these peak13Stuck material in sample line/nozzleFlush sample line with detergent and PBS with increased sample pressure differential (boost).14Contaminating material from previous sampleRun filtered PBS before and after samples, to minimize the presence of contaminating particles.15Coincident detection of particlesSample is too concentratedDilute the sample16Use spike in beads to ensure an operational concentration range17Nanobubbles at the interrogation pointAre created due to the high pressure in the nozzleRun PBS control between samples to have that source of noise identified and controlled