TITLE: Spectradyne nCS1: Sample measurement and device maintenence protocol
AUTHORS: Bryce Killingsworth, Joshua Welsh, Tim Traynor, Michelle Pleet, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a generic protocol for using the Spectradyne nCS1 resestive pulse sensing instrument to measure the diameter distribution and concentration of nanoparticles.</div></div>

[STEPS]
?. [Preparation of running buffer]
If running buffer is more than two weeks old, it must be prepared fresh.
?. [Preparation of running buffer]
Discard the old running buffer contained in the nCS1 buffer bottles.
?. [Preparation of running buffer]
Rinse out the buffer bottles with a small amount of purified/filtered water.
?. [Preparation of running buffer]
If there is no previously prepared 1% Tween-20 solution, prepare a  bottle of 1% Tween-20 by pipetting 5 mL of pure Tween-20 into a new sterile  bottle of DPBS.
500 mL
500 mL
?. [Preparation of running buffer]
Gently invert the bottle.
?. [Preparation of running buffer]
Let the bottle sit for at least 15 minutes while occasionally inverting until Tween-20 is entirely dissolved.
?. [Preparation of running buffer]
Filter half of the Tween-20 solution with a  PES bottle-top filter.
?. [Preparation of running buffer]
Equally divide the filtered  Tween-20 solution into the two nCS1 buffer bottles.
?. [Preparation of running buffer]
Store running buffer at  and mark the date it was prepared so it can be changed in two weeks.
4 °C
?. [Device start-up]
Turn on and log in to attached PC.
?. [Device start-up]
Obtain nCS1 buffer bottles and water bottles from the  fridge.
4 °C
?. [Device start-up]
Attach the nCS1 buffer bottles to their indicated fluid lines on the side of the instrument.
For all bottles, when refilling, be sure to screw the caps on tightly. Loose caps on the buffer bottles may cause pressure leaks that will result in the system from pressurizing and/or preventing chips from priming.
?. [Device start-up]
Turn on the nCS1 with the power switch on the back of the instrument.
?. [Device start-up]
Open the nCS1 software.
?. [Device start-up]
The bar at the top of the software should be green with the word "Ready" displayed. If not, shut off the nCS1, restart the PC and then repeat the power up process, ensuring the software is only opened after the nCS1 is powered on and properly connected via its USB cable.
?. [Device start-up]
If the user determines the device needs to be cleaned, obtain a cleaning cartridge.
We recommend to run the cleaning cartridge at least once when turning on the instrument.
?. [Device start-up]
Properly position cartridge in slot on stage. The blue indicator light and the blue ring light should both illuminate.
?. [Device start-up]
Press the outside button to raise the stage before clicking 'run cleaning cartridge' in the software.
?. [Device start-up]
Wait about three minutes for routine to finish.
?. [Device start-up]
Lower stage by pressing metal button and return cartridge to safe storage place.
?. [Sample preparation and loading ]
Prepare a  solution of Tween-20 in Thermo Fisher Gibco Sterile DPBS (identical to the running buffer) or a  solution of BSA in Thermo Fisher Gibco Sterile DPBS, ensuring ample time given to allow solids to dissolve. PBST (PBS with 1% Tween-20, filtered) should be used unless investigating buffer effects on lipid particles, as it is the default recommended buffer. Researchers with Edwin van der Pol's group have investigated other methods of removing small particles from Spectradyne sample buffers which could be considered.
?. [Sample preparation and loading ]
Filter the solution with a Whatman Anotop 10, 0.02 µm syringe-driven filter into clean plastic tubes.
?. [Sample preparation and loading ]
Select a cartridge type to load your sample into:
?. [Sample preparation and loading ]
Calculate the volumes of filtered  Tween/PBS, spike-in beads, and sample particles to mix together. It is suggested that the final concentation of spike-in beads be 5E9 particles/mL*, the final concentration of sample particles be around 1E10 particles/mL* and that the overall volume is at least  to allow sufficient mixing.For C-300 cartridges use a 203 nm NIST PS spike-in beadFor C-400 cartridges use a 269 nm NIST PS spike-in bead*These values were listed in May 2020 and may need to be updated at a future date based on future experience with a variety of samples
50 µl
?. [Sample preparation and loading ]
Thoroughly vortex spike-in beads (for example, 10 short pulses at max power) and pipette into   Tween/PBS buffer. Vortex the tube thoroughly to avoid any bead aggregates.
?. [Sample preparation and loading ]
Vortex sample particle sample then pipette into the buffer/bead mixture. Vortex well.
?. [Sample preparation and loading ]
Obtain a new sample cartridge and a P10 pipette set to .
4 µl
?. [Sample preparation and loading ]
Vortex sample particle mixture immediatlely before drawing up  of sample and inspecting filled pipette tip to ensure the absence of air bubbles.
4 µl
?. [Sample preparation and loading ]
Using one hand to hold the cartridge in place, insert pipette tip into the fluid loading aperature until it gently contacts the bottom of the loading chamber.
?. [Sample preparation and loading ]
While slowly pulling the pipette tip upward so that it is not contacting any surface load ~ of the sample into the bottom of a sample cartridge, so that a small volume of sample is left in the tip and an air bubble isn't pipetted into the cartridge fluid well.
3.5 µl
?. [Sample preparation and loading ]
Tap the cartridge against the table surface to dislodge any bubbles then hold it at eye level to inspect the fluid level and check for an even meniscus absent of any bubbles. and then insert onto cartridge stage.
?. [Sample preparation and loading ]
If there is a bubble, try alternative ways of tapping the cartridge, or, use the P10 pipette to aspirate the fluid out, several times, and retry.
?. [Sample preparation and loading ]
Load the cartridge onto the stage and ensure both the blue indicator dot and the blue ring light are illuminated.
?. [Running sample on nCS1 device]
Use the nCS1 Cartridge Log document on SharePoint to record detailed information about each sample run on the instrument. This information is critical for identifying measurement quality over time.
This step is specific for the NCI Translational Nanobiology Section's use.We do, however, recommend creating a log of your cartridge and its lot number to track performance over time.
?. [Running sample on nCS1 device]
Enter mold ID and box ID information into the nCS1 software.
?. [Running sample on nCS1 device]
Enter a descriptive prefix for the sample and a sample description if desired.
?. [Running sample on nCS1 device]
Create a new folder on the PC to store mesaurement data with date and descriptive name. Select the new folder as the file location for the measurement.
?. [Running sample on nCS1 device]
Visually confirm the stage is raised so that the cartridge is properly positioned to receive fluidic inputs.
?. [Running sample on nCS1 device]
Press "GO!" to begin automated procedure to prime cartridge. If the instrument has not had a cleaning cartridge run, the first priming procedure will take longer than the subsequent primings. (update as of 7/27/20: if the instrument is failing to prime, press "Stop" and then press "Clear constriction" and then try priming again).
?. [Running sample on nCS1 device]
After priming, the instrument will automatically begin taking 10 second acquisition measurements (also known as to as "traces".)
?. [Running sample on nCS1 device]
The blue line indicates the instrument voltage. Sharp spikes indicated particles pasing through the nanoconstriction and red Xs indicate events that the software determines to be particle clogs of the nanoconstriction. A clog-free trace will automatically be processed by the auto-analysis engine from a raw file into a stats file, which will then be plotted as a "Live CSD." If the number of transient clogs excedes a threshold, the software will not "flag" the raw file from the trace for processing and a stats files will not be automatically generated for that trace.
?. [Running sample on nCS1 device]
Using the auto-analysis engine's live CSD tool, particles within a diameter range can be selected and the concentration   within the range displayed. This tool can be used to determine how long to acquire data, for example, until 10,000 spike-in beads have been measured.
If the live CSD tool does not appear (may happen if the program needs to be restarted due to a crash), just click "Restart Auto-analysis Engine" under the Manual Utilities and be sure to continue processing accumulated files.
?. [Running sample on nCS1 device]
After the user is satisfied with the data collected, press the Stop button to end data collection.
If the instrument clogs, as indicated by the recent acquisitions covered in red Xs and data not saved, click "Stop", "Clear Constriction", and then "Continuous" when allowed. This should fix most clogging situations and combine the data from the previous half of the run.
?. [Running sample on nCS1 device]
Press the End Run button and allow end run sequence to procede. After the sequence is finished the blue ring light should be iluminated.
?. [Running sample on nCS1 device]
Press the metal button, remove and discard the used cartridge.
?. [Running sample on nCS1 device]
Samples can continue to be run on new cartridges as described until done with nCS1 session.
While the next sample is running, it possible to go to the Data Viewer software and combine the files from the previous sample acquisition to save time. To do so, select the correct folder, select all raw files from the past run, bring them down into the lower "currently loaded" box. Click "Process All" under the Process Raw Data tab. This will create stat files from the raw data. For the Translational Nanobiology Section pipeline ensure all filters are removed before clicking 'Combine Data for All Loaded' under the "Data Output" tab.
?. [Device shut down ]
Load a cleaning cartridge onto the stage and raise the stage.
?. [Device shut down ]
Select the "Instrument shut down" button and follow the on-screen instructions.
?. [Device shut down ]
It is recommended to leave the DI water bottles attached to the machine while it is off in order to keep the lines from outside contaminants, and to keep the O-rings from cracking and drying out readily.
?. [Troubleshooting]
AB1ProblemSolution2After stage is lowered fluid is actively dripping onto the cartridge or stageImmediately position a Kimwipe in the cartridge loading area to absorb fluid and turn off power switch on nCS1 which should cause fluidics lines to be clamped shut. Continue monitoring fluid dripping being careful to minimize fluid entering compartments of the device. Do not turn device back on; unplug fluid bottles, use compressed air gently on the electronics of the stage. Allow to dry overnight until turning back on to prevent any electrical shorts.3The blue ring light doesn't illuminate after the completion of an "End Run" proecedurePress CTRL + R to open an additional window of control buttons in the nCS1 software. Make sure that the End Run procedure has actually been completed, if uncertain it can be run twice. Then, check the checkbox next to overide fluid safety control and acknolwedge the sofware's warning. Then, press the "move stage down to limit" button to lower the stage and remove the cartridge. The fluid safety control box can remain checked as long as the user never presses the "move stage down to limit" botton unless they are certain the "End Run" procedure has been completed.4The blue ring light and/or the blue indicator light doesn't illuminate when a cartridge is positioned on the stageThe cartridge can be moved gently back and forth on the stage to make sure it's positioned well. It should be able to easily slide back and forth if it is positioned flat on the stage. If this still doesn't work, the user is certain it is positioned properly and still would like to procede, press CTRL + R to open an additional window of control buttons in the nCS1 software. Then, check the checkbox next to overide fluid safety control and acknolwedge the sofware's warning. Then, press the "move stage up to limit" button to raise the stage. The fluid safety control box can remain checked as long as the user never presses the "move stage down to limit" botton unless they are certain the "End Run" procedure has been completed.5There is an error message saying no electrical connection with cartridgeThis may be caused by dust on the electrodes in the nCS1 that interface with the cartridge. A kimwipe can be used to gently wipe them down, or compressed air.6There is an error message saying connection with device lostThis is almost always caused by the mini USB connector cable being loose. Make sure it is tightened and the instrument may need to be restarted.7The nCS1 loses connection with the cleaning cartridge during instrument shut-down (blue indicator lights turn off)This means that the automated shut-down procedure cannot be completed. Press "End Run" to drain fluid lines and prevent fluid leaks, then use "Stage Down" in the CTRL + R menu, with the fluid safety box checked, to remove cartridge. Remove fluid bottles and turn off instrument, being careful to watch for any fluid that may drip.
AB1ProblemSolution2After stage is lowered fluid is actively dripping onto the cartridge or stageImmediately position a Kimwipe in the cartridge loading area to absorb fluid and turn off power switch on nCS1 which should cause fluidics lines to be clamped shut. Continue monitoring fluid dripping being careful to minimize fluid entering compartments of the device. Do not turn device back on; unplug fluid bottles, use compressed air gently on the electronics of the stage. Allow to dry overnight until turning back on to prevent any electrical shorts.3The blue ring light doesn't illuminate after the completion of an "End Run" proecedurePress CTRL + R to open an additional window of control buttons in the nCS1 software. Make sure that the End Run procedure has actually been completed, if uncertain it can be run twice. Then, check the checkbox next to overide fluid safety control and acknolwedge the sofware's warning. Then, press the "move stage down to limit" button to lower the stage and remove the cartridge. The fluid safety control box can remain checked as long as the user never presses the "move stage down to limit" botton unless they are certain the "End Run" procedure has been completed.4The blue ring light and/or the blue indicator light doesn't illuminate when a cartridge is positioned on the stageThe cartridge can be moved gently back and forth on the stage to make sure it's positioned well. It should be able to easily slide back and forth if it is positioned flat on the stage. If this still doesn't work, the user is certain it is positioned properly and still would like to procede, press CTRL + R to open an additional window of control buttons in the nCS1 software. Then, check the checkbox next to overide fluid safety control and acknolwedge the sofware's warning. Then, press the "move stage up to limit" button to raise the stage. The fluid safety control box can remain checked as long as the user never presses the "move stage down to limit" botton unless they are certain the "End Run" procedure has been completed.5There is an error message saying no electrical connection with cartridgeThis may be caused by dust on the electrodes in the nCS1 that interface with the cartridge. A kimwipe can be used to gently wipe them down, or compressed air.6There is an error message saying connection with device lostThis is almost always caused by the mini USB connector cable being loose. Make sure it is tightened and the instrument may need to be restarted.7The nCS1 loses connection with the cleaning cartridge during instrument shut-down (blue indicator lights turn off)This means that the automated shut-down procedure cannot be completed. Press "End Run" to drain fluid lines and prevent fluid leaks, then use "Stage Down" in the CTRL + R menu, with the fluid safety box checked, to remove cartridge. Remove fluid bottles and turn off instrument, being careful to watch for any fluid that may drip.