LSR II Start-Up Protocol: (Monday
Contents
1. there are different configurations that pair with each nozzle size be sure that you have selected the configuration that matches the nozzle size you are using Please refer to Changing the Nozzle Size amp Configuration in the User Manual If you are on the LSRII and do not require any filter changes for your experiment leave the configuration as is and proceed with CS amp T Grab the QC Beads from the FACS Core fridge look in the white BD box There should already be a 5ml tube inside the QC box containing beads diluted in H20 but if there isn t first vortex a QC bead vial and then add 2 drops per 1ml H20 in a 5ml tube to achieve an optimal concentration Ensure that the bead lot ID listed in the CS amp T software window matches the bead lot ID that is actually listed on the QC bead vial look on the box and the individual vials containing the QC beads They should be the same lot ID as the flow core regularly checks to ensure QC beads are up to date If not please inform the flow core manager and ask for assistance it is not mandatory to run QC before every experiment if you are unable to locate the beads etc proceed with your experiment and simply skip QC Load the 5ml QC bead tube onto the cytometer and click on the green RUN button Ensure that the drop down box above it says Check Performance The CS amp T software will now perform Quality Control which takes 5 7min If there are2. Running CS amp T Beads 1 Open FACSDiva software and ensure that the cytometer Says connected in the lower right corner of the screen 2 From the top menu bar choose Cytometer gt CST for Aria I amp I you must first disable the sweet spot function for the stream 3 CS amp T will take control over the cytometer and a new window opens this is the CS amp T software interface will look similar to below for reference only gt Cytometer Sctup and Tracking Ble tometer Ioas stup Reports Perfomance Trading System Summary RequietAttentor Load a carousel wth te bead tubes ard dick Pur button to tat aup E EPRE Choracteree Check Perfomance Cytcmete Contigeratior 2 lyser S color 4 29 BD cefauk Lor ID 5441 Load Tube Marualy E3 Cytomecr Bastire No Cytometer Bessine svalsble for ourert configuration end dead lot cytometer Corfiguraoon bser amp color 4 2 BD default Lylometer Paillo mance No C ytometer Performance s avalshie Foe curent corfiguraocon and oer Int Select Configuracton y J g Shutdown Souticon Cleaning Sowion Yacuum Heer swta Pump Wiasle Taiz Facsriow Pressure Fac Seow Love Sample Pressure 1 2 3 4 5 6 First you will need to select the configuration that you plan to use for your experiment A configuration is a saved software profile for a particular set of filters and or a nozzle size If you are on the Aria I or II
3. any errors upon completion please notify the core manager The results are saved in the software so you may click OK and proceed with your experiment if the manager is not in the lab Post Experiment Cleaning LSR II After you finish your experiment please perform the following cleaning procedure 1 Load a tube of 10 Bleach and leave the SIP arm to the side for 2min This cleans the SIP arm of any cells debris so they don t enter the system 2 Acquire the tube of 10 Bleach for 5 minutes Clean 3 Load and Acquire a tube of ddH20 for 5 minutes Rinse ti Ei 4 From File tab select Log Out to exit your account Shut Down LSR II If you are the last user of the day please shut down the cytometer and computer 1 Ensure that the cleaning procedure has been performed 2 Exit Diva software and turn off the computer standard windows shutdown 3 Turn OFF the LSR II by pressing the GREEN power button on the front right LSR II Post Experiment Cleaning docx Created by Dave Stanley
4. E WEAR PPE 1 Ensure that the LSR II is in Standby Mode 2 Disconnect the waste supply line orange from the waste tank by pressing on the silver metal clasp Next disconnect the waste tank sensor by twisting the silver connector at the top of the tank counter clockwise until it releases 3 Carry the full waste tank to the Flow Core sink and CAREFULLY dump it out Refill the waste tank with 1 Liter Bleach located under the sink ONLY use the graduated cylinder that says FOR BLEACH ONLY and pour very slowly 4 Return the empty tank with 1L Bleach back to its position under the LSR II bench and reconnect both the orange supply line and the black tank sensor line 5 The waste tank is now ready for operation Quality Control QC When performed on a regular basis QC provides a reliable standard for monitoring a cytometer s performance over time The CS amp T software tracks laser and fluidics performance and makes adjustments to laser delays PMT voltages and area scaling factors to ensure consistent operations from day to day Without regularly running QC user data and or sorting performance may become inconsistent over time It is extremely important to run QC to ensure cytometer accuracy and consistency Cytometer Setup amp Tracking CS amp T e Defines baseline laser performance e Tracks cytometer s overall performance e Establishes and automatically updates application settings
5. LSR II Start Up Protocol Monday Friday left cover side door a right cover fluidics interconnects control panel cytometer handle power switch 1 Turn on the power to the computer attached to right side of the workstation at the Windows login prompt select administrator and enter BDIS as the password to log in and gain access to the computer 2 Push the GREEN POWER SWITCH on the lower right corner to turn on the LSR II 3 ALLOW LASERS TO WARM UP FOR 30MIN BEFORE RUNNING SAMPLES IF YOU ARE THE 1ST USER OF THE DAY AND LSR IS NOT ALREADY ON 4 Double click the BD FACSDiva Software desktop icon to launch program 5 When prompted SELECT YOUR P I as the user and leave password field blank 6 Once the FACSDiva software has connected to the cytometer usually 1 2min it will ask you to Choose CST Settings Select Keep Current Settings If you see the message Cytometer Disconnected after launching the FACSDiva software click on Cytometer from the top menu and select Connect to re connect the software If this is unsuccessful try power cycling the cytometer turn off and on by pressing the green power switch and then click Connect again If this still did not work try power cycling both the workstation computer and the cytometer LSR II Daily Start Up docx Created by Dave Stanley 7 Verify the sheath and waste tank fluid levels If the sheath
6. between samples When the arm is to the side a vacuum is continuously aspirating thru the SIP e Ifasample tube is left on the SIP while the support arm is to the side a vacuum will aspirate the sample into the waste tank Be careful not to leave a sample on the SIP with the arm to the side or you may loose your sample Remove and load samples quickly so as not to loose any cells in the vacuum cleaning Arm to the side vacuum ON Arm centered vacuum OFF LSR I Daily Start Up docx Created by Dave Stanley LSR II Sheath Tank Management gt TO REPLENISH THE SHEATH TANK PLEASE WEAR PPE 1 Locate a new or already open box of BD FACSFlow 20L 2 Press the Standby button on the control panel light turns orange 3 Disconnect the supply and air pressure lines from the steel sheath tank 5 Open the steel sheath tank by twisting the black clamp counter clockwise 6 Push DOWN on the oval shaped lid until the lid drops down into the tank 8 Next pour sheath fluid from the FACSFlow box into the steel sheath tank 9 Fill until the fluid level reaches the upper groove line that is visible inside 10 Re insert the tank lid and tighten with the black clamp until extremely tight The clamp arm must cross the short axis of the lid to create a proper seal 11 Re attach the supply and air pressure lines to the sheath tank 12 Check the sheath filter for air bubbles if there is any air trapped inside
7. in the filter you will need to purge it First flick the filter several times to get any smaller bubbles to accumulate into one big bubble It helps to hold the filter at a 45 angle with the release valve opening pointed up so that the air bubbles accumulate next to the opening Next find a container or dish to catch the fluid that is going to leak from the filter vent tubing during the air purge and hold this dish under the filter vent Then slowly open the roller clamp by sliding the roller towards the tube opening until the air bubble has completely purged from the filter there will be some sheath fluid leaking out after the air bubble so Be Ready Close the roller clamp immediately after all air bubbles have been purged You may need to flick and repeat 13 Locate the second roller clamp at the back right side of the LSR II and slide the roller open same as above to release any additional air trapped in the fluidics Be sure to catch the excess fluid that will come out in a container and close the roller clamp when you are sure that all the air has been purged 14 Press the PRIME button on the front control panel to prime the fluidics system for operation this flushes the flow cell with new sheath fluid The prime should take about 30 seconds and the light will turn RED during the prime and back to GREEN when finished ee ea aw em LSR II Waste Tank Management gt TO EMPTY THE WASTE TANK PLEAS
8. tank level is low you will need to replenish the sheath supply if the waste level is high you will need to empty the waste tank PLEASE WEAR PPE SEE SHEATH TANK MANAGEMENT PROTOCOL TO REFILL SHEATH SEE WASTE TANK MANAGEMENT PROTOCOL TO EMPTY WASTE 8 When you are ready to acquire your first sample PRESS THE RUN BUTTON on the front control panel gt the light will turn from ORANGE standby to GREEN running Pre Experiment Cleaning 9 Load and Acquire a tube of 10 Bleach for 5 minutes Clean 10 Load and Acquire a tube of ddH20 for 5 minutes Rinse a 11 Proceed with your experiment Sample Flow Rates LO 12ul min MED 35ul min HI 60ul min These values are approximate and may be by adjusting the SAMPLE FINE ADJ knob The knob turns 10x times adjusting the flow rate for each setting H M L from 0 5x to 2x the sample flow rate The 5 turn marks the 1x point for each setting which reflects the sample flow rate values listed above LSR I Daily Start Up docx Created by Dave Stanley Basic Cytometer Info The LSR II s Sample Injection Port SIP is where the samples are loaded for analysis outer sleeve sample injection tube Tube support arm e After each sample is unloaded and arm is pushed to the side the LSR II runs a continuous vacuum thru the sample injection tube to backflush any excess fluid and prevent carryover of cells
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