Checking the fluids of the cart

Transcription

1 1 Preparing FACSAria for sterile sorting Prepare and autoclave these fluids at least 2 days in advance: Fluids Remarks Prepare 5L (for 1 period) or 10L (for 2 Each stainless-steel-sheath-tank has a periods) of PBS (Mg- Ca-) in the stainlesssteel-sheath-tank. Place the tank in a biohazard tank, write your details and the tank number written on it. When you take a bag. number in the tanks logbook and Fill the assigned glass container with 500ml document the date of return. DW. Place the container in a biohazard bag. When you have returned the aoutoclaved Prepare two small bottles with 100ml PBS and containers, take 3 stickers and write the 100 ml DW. Place them in a small separate required information. biohazard bag. Do not stick anything directly on containers. Instead, attach stickers to the plastic bags only. Close the lids of the DW and PBS containers after autoclave. Procedure STEPS 1. When computer is off, turn it on and login with: Username - Operator Password public Turn on the FACSAria - green button on the left side of the instrument. Turn on the lasers- white button / mechanical knobs. Turn on the cooling system ("LAUDA") (*beep sound for a couple of seconds is normal). When working with human cells - Turn on the AMO system. Checking the fluids of the cart 2. Check the waste container status. To pass start-up with no problems, the waste tank should be no more than ¾ full. Check Ethanol tank status. Weigh the Ethanol Steel-tank on the electric scales. It should weigh a minimum of 5.4 kilo; if it weighs less add 70% Ethanol. Ready-made 70% Ethanol is on the sink, in 5 L plastic containers. 3. DoubleClick on BD FACSDiva ICON to upload it. Login with your lab s username and password. When a window pops up - choose- "use CST setting" (wait patiently). NOTES Validate that temperature has reached 4 C. It takes minutes.!!! Remember to register in the AMO logbook -----!!! A full waste container should be replaced with a reserve empty one, placed by the side of the cart. Confirm the empty container has 500 ml sodium hypochlorite (EKONOMICA). Empty the full waste tank to the sink and add 500 ml of sodium hypochlorite (that can be found under each sink). Place the empty tank by the cart. A full waste container will stop the stream without warning, giving a clog message. Keep track of the waste status. In general-diva is a heavy & slow program. Be patient. For public use, login with: Username - public Password - public

2 2 4. Place sterile steel Sheath tank on the scales. Scales are used in sterile mode instead of the PBS level sensor. The scales allow tracking the PBS level as you run. 5. Replace sterile DW container as followed: Disconnect the currently connected DW container and replace it with the sterile DW container you prepared. Go to Cytometer cleaning mode prime after tank refill check the DI box O.k. Make sure the filter on top of the DW container is standing straight. Repeat this step until the DW tubing has no air in it. 6. Go to cytometer cleaning mode prepare for aseptic sort, The following instructions will pop. Total sterilization procedure takes 45 minutes Open the sorter cover and follow the steps with the following changes: 7. Ignore this step and press Done 8. No remarks 9. No remarks 10. No remarks Prepare sterile work environment: change diaper on bench. Bring a sterile petri-dish (used as sterile space for tube caps). Prepare 2 FACS tubes, containing: 1. Sterile DW (from the 150ml you prepared) 2. Sterile PBS (from the 150ml you prepared) This step takes 25 minutes. Ignore this step. Follow our remark in line 12. Instead, disconnect the blue line from the side of the cart and connect it Air tube = transparent tube line directly to the sterile sheath tank. Then, connect the transparent (air) line to the sterile tank. Press Done for the coming 2 steps And then press following Fluid/sheath tube = blue tube line Run. 13. Ignore this step and press Done 14. Ignore this step and press Run The software will direct to a new instructions-window of Fluidic start-up %

3 3 16. Follow the steps directed by the software. Pay attention to the remarks written for each step, in this protocol. After each step you performed press done Fluidic startup 17. For your safety, first connect the fluid line and only second the air line. 18. Don t be tempted skip this step While fluidic startup is running, check the nozzle you intend to use under the microscope. If it is not clean, sonicate it in 70% ethanol for 1 minute and air dry. Sometimes this step has to be repeated several times until the nozzle is clean. Always validate that the nozzle is clean under the microscope. After you remove the closed nozzle loop and before you insert the required nozzle validate that all the slots, sockets, camera area and deflection plates are dry This step takes ~5 minutes. When it reaches 30%, Air flow will enter the sheath tank- At this point fix air leaks, if any. For high speed sorting use 70μm nozzle For medium speed 85μm For low speed 100μm For extra low speed 130μm

4 4 Stream operation 1. Validate configuration. In the stream window you can see configuration set up (picture 1a). If there is a mismatch between the nozzle you inserted and the configuration set-up, go to: 1) Cytometer view configuration (if a configuration window doesn t appear, minimize the DIVA, you ll find it behind) 2) Choose the correct nozzle configuration 3) Press set configuration a message window pops up: "make sure the filters and mirrors " 4) Press Ok. 5) Press Ok again at the right side of the window. 6) Close this window at the windows X Picture 1 Stream window a. Speed configuration b. Turn on stream by clicking the red x 7) Choose "use CST settings" in the pop up window Turn on the stream by clicking the red X in the stream window (picture 1b). Wait until it changes to green.

5 5 Centering the stream 2. ***Attention*** beware!!! Don t touch the deflection plates when high voltage is ON. Open the deflection plates door. The stream between the two deflection plates should hit the center of a narrow tab (waste drawer). If not, center it by first loosening two adjustment screws and then manually moving the sort box to center position. Re-tighten the screws to lock the position of the box. To handle the screws use an Allen type screwdriver found in the first drawer near each Aria desktop (yellow handle). When the stream is centered Close the deflection plate s door. Close the sorter cover Flow cell Adjustment screws Deflection plates AccuDrop laser adjust (silver knob) Wa ste drawer Narrow Waste tab 3. Setting the sweet spot Setting drop 1.Drop1 is the breaking point of the steam to drops and it is controlled by the amplitude. Two fields represent drop1 values: the left shows the last saved value. The right shows current value. Current value will change when the amplitude is changed. You should bring the right and left fields values to as close as possible. It is achieved by scrolling up or down the amplitude. It is not required to reach the exact value but to find the closest value today. Once you got the values close, set the gap Setting the gap - fine tune the amplitude so that the Gap right value matches the left Gap value. Once the gap values match (+-2) go to Drop 1 left field (last saved value) and type in it the value you got on the Drop1 right field (current). Activate the sweet spot to keep the stream conditions stable. Picture 3 Sweet spot ICON Drop Breaking point Frequency value Typical for 70 nozzle Last saved value Drop 1 and Gap Amplitude scroll Current value Drop 1 and Gap

6 6 Calibrations: Calibrations can take a few minutes if the system is clean and up to an hour if you find out it needs cleaning. Consider it in your time planning. Rainbow Calibration Rainbow beads (RB) are used to check the system performance and that it is sufficiently clean You should find a ready to use tube in the refrigerator. The tube is marked RB and dated. To prepare a new RB tube for calibration, transfer 2 drops of RB from the stock vial into ~0.5ml DW a) In the Browser window open Shared view, DoubleClick on the notebook named Rainbow or RB (it should turn into an open notebook. If a window pops up, choose continue ). b) Open the specimen of the current month. Insert a new tube and name it with the current day. Activate the new tube (activated tube turns from gray to green). c) Go to cytometer cleaning modes sample line backflush start ; let it wash the sample line for a few seconds. Press Stop and cancel. d) Load a tube containing DW and let it run for 30 seconds. (This step is essential to wash residues from the sample line that may destroy the APC signal of the RainBow). e) Validate that all the lasers are turned ON f) Vortex & Load a tube with RainBow beads g) Acquire at the preferred speed of no more than 400 evt/sec h) Press Record i) Unload the RB tube j) Confirm that P1 is more than 65% k) Confirm that each one of the histograms has 8 peaks l) Confirm that the CV of the last right peak is no more than 5% If RB results are satisfactory, proceed to AccuDrop calibration. Do not proceed to AccuDrop calibration if RB calibration is faulty.

7 7 AccuDrop Calibration AccuDrop beads (AC or AD) are used to set the optimal drop delay for sorting. You will find a ready to use tube in the refrigerator marked AC and dated. A fresh tube can be made by adding 1 drop of AC from the bottle to ~ 0.5 ml DW Steps 1) Go to Shared view Open AccuDrop /AC experiment; Do so by DoubleClicking on the notebook. If a window pops up, choose continue. 2) Press the + of folder Global worksheet' 3) Press + of page Global sheet1' and DoubleClick Sort layout 4) Confirm the sort layout conditions: Tube device: 4 tubes. Precision: FINE TUNE. Target events: Continuous. The gate NOT P1 should be set in the left stream position Notes 5) Press the + of the specimen and activate the tube. 6) Click on voltage (turns red) in the sorting window press test sort icon. Four side streams should appear. The location of the side streams can be adjusted if needed, with their sliders. Close side streams by pressing again test sort & voltage. 7) Vortex & Load AC tube Picture 4 Sort window Main stream Image Assure to have a fit range of events/second: For 70 micron = 1,000 to 3,000 evt/s For 85 micron = 800 to 2,000 evt/s Side streams sliders use them to reposition side streams For 100 micron = 600 to 1,500 evt/s For 130 micron = 400 to 1,200 evt/s If evt/sec is lower than desired rate, increase the flow rate, you can go up as much as flow rate-5 on the other end, if beads are too concentrated dilute them with DW/PBS. 8) Press Sort Choose cancel. a. Turn on the voltage; a side stream When you choose cancel beads are sorted and delivered to the waste.

8 8 appears on the left Confirm that the Image of the main stream and left-side streams in the sort window are focused. Do so by turning the AccuDrop laser adjust (Picture 2, silver knob) either clockwise or counterclockwise until the two streams are brightest/strongest. b. When streams are focused, press on optical filter icon. c. Two squares appear. Each stream should be inside the each square. d. Confirm that the right & left % of beads make ~100% altogether. e. Press AutoDelay in the sorting window. You can also do manual setting of the drop delay; in the sorting window, gradually increase or decrease the drop delay by clicking it s up/down arrows. Choose the drop delay that yields % of the events in the left square. For each change you make in the drop delay wait ~2 seconds to let the sorting values stabilize. 9) Unload AC tube Run RB whenever: 1) Following start-up procedure. 2) If you changed a nozzle. 3) As part of troubleshooting step. Run AC whenever: 1) Following start-up and only after RB test passed. 2) When changing a nozzle. 3) When Drop 1 has changed in more than 20 units. 4) If you performed clean the flow-cell protocol. The system is now ready for sterile use