Moflo Procedures Startup 1. Turn on recirculating temperature bath for sample cooling (4 °C) a. Turn both switches ON. b. Press Set Button ( ) once (should see F1). 2. Add bleach to the waste tank and close it tightly. 3. Close air valve from sheath tank. 4. Connect waste and sheath tank to the respective tube connectors. 5. Start the vacuum pump (switch located on the electric board on the wall). 6. Open the main pressure (BLUE switch) and main vacuum (RED switch) in the pressure console. Wait a few seconds until pressure builds up in the sheath tank. 7. Turn ‘TO FILTER’ (red) switch to ‘PBS’ position. 8. Bleed sheath tank and tubing by opening and closing the metal valve to the position ‘bleed’. 9. Bleed PBS filter by opening and closing the (black) filter valve until no air bubbles are seen. 10. Turn ‘TO SORTER’ (red) switch to ‘PBS’ position. 11. It’s a good time to turn lasers ON in standby mode (Fans start working while lasers are in standby) 12. Turn ON TV monitor, Oscilloscopes and PMTs. 13. Open the two main switches in the sample station to ‘Sheath’ position. 14. Turn ON Drop Drive Amplitude. 15. In the sample station, turn the first switch to ‘Vacuum’ for 10sec. Then switch it between ‘Vacuum’ and ‘Sheath’ positions to make sure drops remain stable and in the same position. Repeat procedure with the second switch. 16. Align stream (check detailed procedures). 17. Align lasers (check detailed procedures). 18. Optimize stream quality and Drop Breakoff position (check detailed procedures). 19. Determine Drop Delay (check detailed procedures). Shutdown 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Switch OFF recirculating water bath (Two black buttons). Set sort plate in ‘Park’ position. Place container beneath central waste tube. Close laser shutters. Switch lasers off (check detailed procedures for each laser). Turn plate voltage OFF. Turn Drop Drive (DD) Amplitude OFF. Remove plates and clean gently with ethanol. Switch OFF stream (Turn main switches in the sample station to the OFF position) Clean sample station and central waste tube with DI water while vacuum is still on. Switch main pressure and main vacuum OFF (red and blue switches on the side of the pressure console). Switch ‘TO SORTER’ and ‘TO FILTER’ valves to the OFF position. Open the sheath tank pressure valve to release air. Switch OFF TV’s and oscilloscopes. Set all PMTs to minimum (400 V) and switch OFF (the most right buttons on both PMT racks) Set all detectors to linear amplification. Disconnect and remove sheath tank, refill it with PBS. Leave tank overnight with pressure valve open to degas. Disconnect and remove waste tank, empty waste carefully and clean it twice with tap water. Leave overnight to dry. Switch vacuum pump OFF (switch located on the electric board on the wall). Finally, make sure that the following is OFF: a. Moflo lasers b. Laser in the culture room. c. Recirculating temperature bath. d. Plate voltage and DD Amplitude. e. Vacuum pump. Align Stream 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Make sure DD Amplitude is OFF. Close nozzle chamber door. Switch nozzle chamber light ON (located on the side of the pressure console). Switch to ‘Video B’ on the main TV (you should see the 3 pinholes). With nozzle in DOWN position: a. Adjust left-right position to opposite side and then re-adjust left-right angle to center stream. b. Adjust backward-forward position to opposite stream border thickness and then re-adjust backward-forward angle to obtain appropriate stream border thickness. Raise nozzle until micrometer leaves the screw (should feel a click). Lower it slightly back to the screw. With nozzle in UP position: a. Adjust left-right angle to opposite side and then re-adjust left-right position to center stream. b. Adjust backward-forward angle to opposite stream border thickness and then re-adjust backward-forward position to obtain the same stream border thickness as in the DOWN position. Lower nozzle to DOWN position and check if stream is aligned. Repeat adjustments in the DOWN and UP position sequentially, until stream is aligned. When stream is aligned, leave nozzle in the DOWN position. Switch DD Amplitude back ON. Switch back to ‘Video A’ on the main TV (you should see the drops). Align Lasers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. y-FL2 y-SSC 14. Turn lasers ON (check specific instructions for each laser). Make sure DD Amplitude is OFF. If not already open, launch Summit software and open ‘Laser Calibration’ protocol. Make sure all detector channels are set to linear amplification. Set FSC gain to 4, and SSC gain to 8. Set CRT1 x and y channels to 1 and 2, respectively (x-FSC and y-SSC). Set CRT2 x and y channels to 3 and 4, respectively (x-FL1 and y-FL2). Open BLUE (488 nm) laser shutter. Place tube with Flow Check beads in sample station: a. Close yellow sample valve. b. Place tube in sample station. c. Lift lever and open red pressure valve. d. Open yellow sample valve. Press boost and/or increase sample pressure until events are seen in CRTs. Adjust sample pressure to obtain 200 events/sec. Start Acquisition in Summit. Align blue laser until signals in all channels fall into the defined regions: a. Adjust left-right and backward-forward stream positions until optimal signal. b. Adjust focus (x-plane), side position (y-plane) and height (z-plane) of FSC lens to maximize FSC intensity. c. Adjust x-y-z planes of BLUE laser lens to increase FL1 and FL2 intensities. d. Readjust left-right and backward-forward stream positions until maximal signal and lowest CV is obtained for FL1 and FL2. When aligned, you should see the following pictures in the CRTs: x-FSC 15. 16. 17. 18. x-FL1 Switch blue laser OFF (Close LASER1 shutter). Close nozzle chamber door. Switch nozzle chamber light ON (located on the side of the pressure console). Adjust the nozzle’s position such that its tip is in the middle of the pinhole that you want the light of your secondary laser to go in to (2nd or 3rd pinhole). 19. Open LASER2 or LASER3 shutter depending on the laser you want to align. 20. Adjust height (z-plane) of the laser you want to align until it hits the nozzle’s tip (you should see the nozzle “lighting” up). y-FL# 21. Lift the nozzle back to its normal position (above the 3 pinholes), and adjust the side position (y-plane) until you see the laser light intercepting the stream (you should see a line reflected on the chamber’s wall). 22. Set CRT2 x channel to 2 (x-SSC), and CRT2 y channel to the respective channel number of the PMT that will be used to align the laser. 23. Remember that most beads need logarithmic amplification, so set the respective detector(s) with the according amplification. 24. Run the calibration beads for the respective laser. 25. Adjust focus (x-plane) and side position (y-plane) of LASER2/3 to increase signal intensity. Every time a local maximum is attained, adjust the height (z-plane) to make sure laser is entering the middle of the pinhole, thus increasing detection intensity. CRT2 monitor should look like this: x-SSC 26. On the digital oscilloscope, adjust the vertical lines to measure the distance between both peaks (Press Run/Stop if needed). The time delay is given on the top-right corner of the oscilloscope monitor. 27. If LASER2 is being aligned, set the value measured in the oscilloscope on the electronic rack, below the trigger button (if value is, for instance, 1.35 s, set it to 1.4 s. If it is <1.35 the set it to 1.3 s). If LASER3 is being aligned, divide the value measured in the oscilloscope by 2 and set it on the electronic rack. Optimize stream quality and Drop Break-off position 1. Make sure DD Amplitude is ON. Make sure strobe is ON and is set to “Change”, so that the phase of drop formation is associated to the strobe phase. 2. Adjust Frequency of drop formation such that the break-off point is as high (closest to the laser interception point) as possible. You might have to adjust camera position (black wheel on the right side of the collection chamber. 3. Adjust DD Amplitude so that the first satellite drop formed is almost connected to the last attached drop. 4. Select 2 Drops. 5. Turn ON both left and right. 6. Turn Plates ON Break-off position 7. Press Test Sort 8. Adjust DeFanning by looking at the middle stream, making Gap sure it’s as thin as possible. Make sure that during this process the drop break-off point is maintained (same position of last attached drop and same gap between last attached drop and first satellite drop). If not, adjust DD Amplitude. Determine Drop Delay Cyclone Drop Delay 1. Insert and run Flow Check Beads in the sample unit. Acquire a few events without recording. 2. Create a small region (R1) around bead population in a FSC x SSC plot. Create a second region (R2) around singlet beads in a FSC x Pulse Width plot. 3. Create a new Sort Decision and set it to sort “R1 & R2”. 4. Run Cyclone in Summit (Sort→Cyclone). Remove any object inside the sort chamber as Cyclone tray will come out. 5. Place slide on Cyclone tray. 6. Adjust DD Amplitude so that gap between last attached drop and first satellite drop is minimal. 7. Start sort in the Cyclone window, running beads at 200-400 events/second. Cyclone tests 10 different drop delays sorting 160 beads for each value. The 10 drop delays will go from 5 drops below the displayed value on the CSU to 4 above. 8. Count beads in each drop (corresponding to a different drop delay) using a simple fluorescent microscope. 9. In the Cyclone window, right-click on the drop with most beads, and select “most beads”. Then right-click on the drop next to it that had the remaining beads, and insert the number of beads in that drop. th Cyclone should adjust the drop delay so that the 6 drop corresponds to the drop delay that yields the highest number of beads. Fine adjustments may still be needed. 10. Start sort again, and verify if drop delay is correct. th th If there are >4 beads in the 5 or 7 drop, adjust drop delay by selecting the respective drop and inserting the number of remaining beads, and run sort again. Repeat this until there are <4 th th beads in the 5 or 7 drop. Fast Drop Delay Determination (FD3) Option 1. Create a small region in the top right corner of a FSC x SSC plot. Name it Drop Delay. 2. Create a new Sort Decision and set it to sort “NOT Drop Delay”. This will sort all the events that are not within the Drop Delay region. 3. 4. 5. 6. Insert and run Flow Check Beads in the sample unit. Start Sort in Summit. Adjust event rate to >800 beads/second. Adjust DD Amplitude so that gap between last attached drop and first satellite drop is minimal. 7. Adjust laser entering the sort chamber so that it intercepts both center stream and deflected stream. 8. Turn optical filter down. This will eliminate reflection from the laser, and only fluorescence from beads is visible. 9. Once all adjustments are stable, adjust Delay, until most of the fluorescence is visible in the deflected stream and/or most of the fluorescence disappears from the center stream. Adjust Delay above and below this point to make sure it is the correct value. Laser Operation Sapphire-200mW 488 nm Blue Laser Startup: 1. 2. 3. 4. Switch main power button ON. Leave >2 min before initializing laser. Turn key to ON position. Wait a few seconds until power reaches 11%, and yellow led lights up indicating laser is READY. 5. Increase laser power to 70%. 6. Open shutter located on the actual laser. Shutdown: 1. 2. 3. 4. 5. Close shutter, located on the actual laser. Decrease laser power to minimum (11%). Yellow led lights up indicating laser is READY. Turn key to STANDBY position. Display should indicate 3% laser power. Wait >5min. Switch main power button OFF. Spectraphysics 35 mW HeNe 635nm Red Laser Startup: 1. Turn LASER 2 shutter ON. In fact, the shutter switch is connected directly to the laser, turning it on. Shutdown: 1. Turn LASER 2 shutter OFF. In fact, the shutter switch is connected directly to the laser, turning it off. Crystalaser-50mW 561 nm Yellow Laser Startup: 1. Switch main power button ON. Wait a couple of minutes. 2. Turn key to ON position. 3. Leave >30 min until laser power has reached maximum. To measure laser output, manual shutter in the actual laser must be open. Coupled through fiber optics, maximum power should be 36-38mW. 4. Open LASER 3 (or LASER 2) shutter in the pressure console. Shutdown: 1. 2. 3. 4. Close LASER 3 (or LASER 2) shutter in the pressure console. Turn key in laser’s power supply to OFF position. Wait >5min. Switch main power button OFF (in the back of power supply). Oxxyus-50mW 561 nm Yellow Laser Startup: 5. 6. 7. 8. 9. 10. 11. Switch main power button ON. Turn key to ON position. Leave >2 min before initializing laser. Double click ‘Yellow Laser.exe’ on the Desktop. Click ‘Laser’ button. Wait ~5min for laser to initialize. Check status in the drop down menu. When laser status is ON, open laser shutter, by clicking the ‘Shutter’ button. Shutdown: 5. 6. 7. 8. 9. Close laser shutter, by clicking ‘Shutter’ button. Switch laser OFF, by clicking ‘Laser’ button. Wait >5min. Turn key to OFF position. Switch main power button OFF. Innova 90C Laser Coming soon.