 |
| Image Source: BD Biosciences, BD Influx Cell Sorter |
>Disconnect the Vacuum Pump and Fluidic Lines.
>Open Waste Tank and Add 1% Bleach and discard.
>Close the lid tightly (To ensure vacuum build up).
>Reconnect the vacuum Pump and Fluidics Lines.
>Open Waste Tank and Add 1% Bleach and discard.
>Close the lid tightly (To ensure vacuum build up).
>Reconnect the vacuum Pump and Fluidics Lines.
Sheath Tank:
>Release pressure using pressure knob.
>Disconnect all the fluid lines connected to the sheath Tank.
>Open and check the sheath level (if required, add sheath fluid to the sheath tank).
>Close and connect the air and fluid lines.
> Connect Sheath Line to Filter to Instrument.
>Disconnect all the fluid lines connected to the sheath Tank.
>Open and check the sheath level (if required, add sheath fluid to the sheath tank).
>Close and connect the air and fluid lines.
> Connect Sheath Line to Filter to Instrument.
Switching ON:
>Switch on Condenser, Vacuum Pump, Mains of the Instrument and Computer.
>Wait for around 10 minutes to build the pressure in the waste tank and sheath tank.
>Wait for 10 minutes for lasers to fully warm up and turn them ON one by one.
>Make Sure, the displayed sheath pressure matches the pressure build scale on the sheath tank.
>Attach the nozzle to the nozzle assembly and place waste bucket below the nozzle.
>Press “RINSE” and leave it on for 1-2 minutes.
>Press “PURGE” and observe the air inside the fluidics lines and place the debubble reservoir on the waste bucket, add DI water to the reservoir. (Make Sure Nozzle is dipped inside the water).
>Observe the fluid and air crossing the valves. And if there are bubbles stuck and then press “PULSE” to remove those bubbles.
>Observe the fluid and air crossing the valves again and repeat until there are no bubbles.
>Make Sure there are no air bubbles inside the fluidic lines.
>Press “RINSE”, take out the reservoir and waste bucket.
>Press “RUN” and Press “BACKFLUSH” and let the sheath come out of the sample line. (BACKFLUSH for at least 5 minutes).
>Turn on the cytometer interface and the computer.. Turn on the monitors above the computer one by one and press “ILLUM”.
(Pinhole Camera, Droplet Camera and Stream Camera).
>Wait for 5 minutes for computer to boot up properly and click on BDFACS Sortware Software and Click Connect. (You should be able to observe the movement of the tray).
>Click on Restore Workspace and restore the previous or saved QC workspace.. and adjust the drop frequency and amplitude according the desired or set Sheath Pressure.
>Wait for around 10 minutes to build the pressure in the waste tank and sheath tank.
>Wait for 10 minutes for lasers to fully warm up and turn them ON one by one.
>Make Sure, the displayed sheath pressure matches the pressure build scale on the sheath tank.
>Attach the nozzle to the nozzle assembly and place waste bucket below the nozzle.
>Press “RINSE” and leave it on for 1-2 minutes.
>Press “PURGE” and observe the air inside the fluidics lines and place the debubble reservoir on the waste bucket, add DI water to the reservoir. (Make Sure Nozzle is dipped inside the water).
>Observe the fluid and air crossing the valves. And if there are bubbles stuck and then press “PULSE” to remove those bubbles.
>Observe the fluid and air crossing the valves again and repeat until there are no bubbles.
>Make Sure there are no air bubbles inside the fluidic lines.
>Press “RINSE”, take out the reservoir and waste bucket.
>Press “RUN” and Press “BACKFLUSH” and let the sheath come out of the sample line. (BACKFLUSH for at least 5 minutes).
>Turn on the cytometer interface and the computer.. Turn on the monitors above the computer one by one and press “ILLUM”.
(Pinhole Camera, Droplet Camera and Stream Camera).
>Wait for 5 minutes for computer to boot up properly and click on BDFACS Sortware Software and Click Connect. (You should be able to observe the movement of the tray).
>Click on Restore Workspace and restore the previous or saved QC workspace.. and adjust the drop frequency and amplitude according the desired or set Sheath Pressure.
Course Adjustment:
>Stream to Waste: Adjust the stream to the waste using the black knobs (front and right) on the nozzle assembly until full brightness spot. (Stream exactly to the waste).
>Stream to Pinhole: use the focus knob and left & Right Silver knobs (Video 1) to the align the stream to the pinholes.
>Lasers to Pinholes: Close the Front shutter and open shutter of the lasers. Place a finger on the sensor to activate the laser. To align the light beam to 1st Pinhole, adjust the horizontal knob of the blue laser, while viewing pinhole video, such that a bright line of light is seen on the 1st Pinhole. (Primary laser to the 1st pinhole). Repeat for the other lasers and align them to their respective pinholes.
>Stream to Pinhole: use the focus knob and left & Right Silver knobs (Video 1) to the align the stream to the pinholes.
>Lasers to Pinholes: Close the Front shutter and open shutter of the lasers. Place a finger on the sensor to activate the laser. To align the light beam to 1st Pinhole, adjust the horizontal knob of the blue laser, while viewing pinhole video, such that a bright line of light is seen on the 1st Pinhole. (Primary laser to the 1st pinhole). Repeat for the other lasers and align them to their respective pinholes.
Alignment and QC:
>Differential Pressure Set up: For example, If for 100 micron nozzle desired sheath pressure is 17 psi then, first using the sheath pressure knob adjust the pressure to 17psi and using the Sample Offset knob decrease the pressure as much as you can and then using sample knob set the pressure to 14psi and then with Sample Offset knob increase the sample pressure up to 20psi…
and then press and hold the boost button and using sample boost set it to 24psi.
Now, sheath pressure is set up 17 psi and sample pressure to 20psi. Usually for all the experiments and others, you can keep the sample pressure to 18psi. Increase or decrease using sample offset depending on the events rate per second desired or required.
>Take 3mL DI water in 5-mL polypropylene Tube and Run water for at least 5 minutes to clean the sample line. And back flush for at least 2 minutes.
>Take 1mL of sheath fluid filtered in 5ml-polypropylene tube and add 1 drop of Rainbow QC beads and mix well by vortexing.
>Place a Tube with QC beads at the sample injection port and take care not to bend the sample injection probe. And secure the sample lock. And Press “SAMPLE”.
>In software, restore the QC workspace including laser delays.
>Select a folder, assign the name, date and delete the existing gate and display count to 500.
>Click on “Acquire” and first set the voltages of FSC and SSC in SSC vs FSC plot… Once data starts appearing, use the focus knobs and adjust the data using PMT voltages and get the maximum (Right Top Corner of the plot). Make sure event rate is between 150 to 250 events per second.
> Once the above step is don, use the horizontal knob to get the maximum right top and clustered population on the plot.
> Adjust the voltage to get the population at left mid. Create a gate on population of interest. If necessary, use primary laser knobs to get the good signal in fsc and ssc.
>Select the gate and apply it to all the histogram plots.
> Do not touch the focus knob, or voltage or stream after the above step for fsc and ssc.
>First adjust the 488nm Blue Laser using Horizontal and vertical knobs and observe the sharp peak and CV.. (CV should be below 6).
>Once the blue laser is aligned to 1st pinhole, align other lasers one by one and adjust the laser delays as well.
>Once all the lasers all are aligned to their respective pinhole and histogram plots have sharp peaks then record the data for 5000 or 10000 events and save the pdf file for the reference.
>Close the QC Experiment.
and then press and hold the boost button and using sample boost set it to 24psi.
Now, sheath pressure is set up 17 psi and sample pressure to 20psi. Usually for all the experiments and others, you can keep the sample pressure to 18psi. Increase or decrease using sample offset depending on the events rate per second desired or required.
>Take 3mL DI water in 5-mL polypropylene Tube and Run water for at least 5 minutes to clean the sample line. And back flush for at least 2 minutes.
>Take 1mL of sheath fluid filtered in 5ml-polypropylene tube and add 1 drop of Rainbow QC beads and mix well by vortexing.
>Place a Tube with QC beads at the sample injection port and take care not to bend the sample injection probe. And secure the sample lock. And Press “SAMPLE”.
>In software, restore the QC workspace including laser delays.
>Select a folder, assign the name, date and delete the existing gate and display count to 500.
>Click on “Acquire” and first set the voltages of FSC and SSC in SSC vs FSC plot… Once data starts appearing, use the focus knobs and adjust the data using PMT voltages and get the maximum (Right Top Corner of the plot). Make sure event rate is between 150 to 250 events per second.
> Once the above step is don, use the horizontal knob to get the maximum right top and clustered population on the plot.
> Adjust the voltage to get the population at left mid. Create a gate on population of interest. If necessary, use primary laser knobs to get the good signal in fsc and ssc.
>Select the gate and apply it to all the histogram plots.
> Do not touch the focus knob, or voltage or stream after the above step for fsc and ssc.
>First adjust the 488nm Blue Laser using Horizontal and vertical knobs and observe the sharp peak and CV.. (CV should be below 6).
>Once the blue laser is aligned to 1st pinhole, align other lasers one by one and adjust the laser delays as well.
>Once all the lasers all are aligned to their respective pinhole and histogram plots have sharp peaks then record the data for 5000 or 10000 events and save the pdf file for the reference.
>Close the QC Experiment.
Accudrop and Sort Set Up:
>Take 1ml of filtered sheath fluid in 5ml polypropylene tube and add 1 drop of accudrop beads. Mix well. Use vortexer if necessary.
>Open a new Workspace and select plots: SSC vs FSC dot plot and FSC histogram plot.
>Create a gate which should select all the data inside the plot. (Gate can be created on dot plot or histogram plot.
>Open the sort set up window and adjust the drop frequency and amplitude based on the sheath pressure and make sure, droplets are properly formed and not changing.
>Using drop charge, drop deflection and stream focus, adjust the drop deflection properly.
>Go to sort window and select Accudrop. And assign the gate for sorting.
>Run the accudrop beads with events rate from 1500 to 2500 events per second.
>Click on Start in sort window and turn accudrop filter to left..
>Adjust the drop delay until all the beads are being sorted to the left.. Very minimum drops with beads to waste.
>Once all the selected beads are being sorted to the left, Turn the accudrop filter back to its original position.
>Record the accudrop data and save sort report for the reference.
>Open a new Workspace and select plots: SSC vs FSC dot plot and FSC histogram plot.
>Create a gate which should select all the data inside the plot. (Gate can be created on dot plot or histogram plot.
>Open the sort set up window and adjust the drop frequency and amplitude based on the sheath pressure and make sure, droplets are properly formed and not changing.
>Using drop charge, drop deflection and stream focus, adjust the drop deflection properly.
>Go to sort window and select Accudrop. And assign the gate for sorting.
>Run the accudrop beads with events rate from 1500 to 2500 events per second.
>Click on Start in sort window and turn accudrop filter to left..
>Adjust the drop delay until all the beads are being sorted to the left.. Very minimum drops with beads to waste.
>Once all the selected beads are being sorted to the left, Turn the accudrop filter back to its original position.
>Record the accudrop data and save sort report for the reference.
Sorter Shut Down:
>Backflush for at least 5 minutes.
>Run 1% facs clean solution for 5 minutes and backflush for 2 minutes after that.
>Run DI water for 10 minutes and backflush for 2-5 minutes after that.
>Close the software and close all the shutters of the lasers.
>Place a waste bucket below the nozzle.
>Stop “RUN” and “BACKFLUSH”.
>Connect the Air Line to Sheath line (Disconnect the connection between sheath filter). Connect it to Sheath Line to Bypass Sheath filter.
>Press “RINSE” and BackFlush and leave it for 5 minutes. And turn off.
>Place the debubble reservoir and add DI Water and press PURGE, let the nozzle take the DI water. (Wait until water is reached the valve).
>Press RINSE and backflush.
>Once all the fluid is removed from the lines, press RINSE to off.
>Repeat this for at least 2 more times.
>Make sure there are no bubbles or fluid present in the fluidics lines.
>Remove waste bucket. Turn off the computer and cytometer interface.
>Remove the nozzle and place it in DI water until next use.
> Turn off all the lasers one by one.
>Turn off the cytometer and air supply.
>Turn off all the main power supply.
>Discard the waste at the end of day.
>Run 1% facs clean solution for 5 minutes and backflush for 2 minutes after that.
>Run DI water for 10 minutes and backflush for 2-5 minutes after that.
>Close the software and close all the shutters of the lasers.
>Place a waste bucket below the nozzle.
>Stop “RUN” and “BACKFLUSH”.
>Connect the Air Line to Sheath line (Disconnect the connection between sheath filter). Connect it to Sheath Line to Bypass Sheath filter.
>Press “RINSE” and BackFlush and leave it for 5 minutes. And turn off.
>Place the debubble reservoir and add DI Water and press PURGE, let the nozzle take the DI water. (Wait until water is reached the valve).
>Press RINSE and backflush.
>Once all the fluid is removed from the lines, press RINSE to off.
>Repeat this for at least 2 more times.
>Make sure there are no bubbles or fluid present in the fluidics lines.
>Remove waste bucket. Turn off the computer and cytometer interface.
>Remove the nozzle and place it in DI water until next use.
> Turn off all the lasers one by one.
>Turn off the cytometer and air supply.
>Turn off all the main power supply.
>Discard the waste at the end of day.
Comments
Post a Comment