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| Cytek Time Zero module user Guide for BD Benchtop Cytometers |
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Article Details
Last Updated
30th of April, 2009
Author
Eric Chase
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CYTEK DEVELOPMENT
TIME ZERO USER'S GUIDE
FACSCAN/FACSORT/FACSCALIBUR/LSR
REV 6/15/03
Introduction
The Time Zero System provides for the rapid addition of reagents to a sample, and rapid introduction of the reagent/sample mixture to the sensing region of the FACScan, FACSort, FACSCalibur or LSR. The module provides sample temperature control, adjustable sample boost time, and variable speed mixing. The module also provides an electronic Time Mark to identify the beginning of the time scale.
The Air Module provides the air supplies required by the Time Zero System.
Electronics Module Controls Description
There are four controls, four connectors, and one readout on the front of the Electronics Module, as shown in Figure 1.
Mixer Off/On: This white push button turns the mixer off and on.
Mixer Speed: This knob controls the mixing speed of the mixing flea.
Boost _Button: This red button activates the sample boost and the Time Mark output to the cytometer's fluorescence 1 channel.
Boost Time: This knob controls the length of the boost time. If the knob is turned fully counter clockwise, the boost is not activated when the Boost Button is pressed; however, the Time Mark is still activated.
Sample Air: This connector provides boost pressure and running pressure to the Time Zero Sample Module. When the air line to the Time Zero Sample Module is disconnected, the air supply is automatically blocked at the bulkhead.
Sample Pinch Valve Air: This connector provides air pressure to operate the sample line pinch valve on the Time Zero Sample Module. When activated, this valve stops backflushing through the Time Zero Sample Line. When the air line to the Time Zero Sample Module is disconnected, the air supply is automatically blocked at the bulkhead.
Circulating Water: These two connectors provide water to the Time Zero Module for temperature control. When the water lines to the Time Zero Sample Module are disconnected, the water supply is automatically shut off at the bulkhead.
Ethylene glycol (antifreeze) should not be circulated through the Time Zero Sample module. The plastic may develop stress cracks from exposure to this material.
Temperature Readout: This indicates the temperature in the Time Zero Sample Module water jacket.
There are two controls and two connectors on the rear of the Electronics Module, as shown in Figure 2.
Figure 2
Power Switch: The Electronics Module is turned on with this.
Time Mark Switch: This sets the length of the Time Mark on the Fluorescence 1 output to either 0.1 second or 1.0 second. The shorter time is used if the software's time resolution is less than 0.25 seconds.
Sample Module Connector: This connector provides power to the mixer in the Sample Module, and connects to the temperature sensor in the Sample Module.
Output BNC: This connector provides the output for the time mark. It is connected to the "X" connector on the rear of the FACScan, or to the FL1 PMT Tee inside the FACSort and FACSCalibur. It is connected to the preamp board on the LSR.
Fuse Holder: The Electronics Module uses a .25 A fuse for 117 Volt operation, and a .12 A fuse for 220-240 Volt operation.
Sample Module Controls Description
There are two controls on the Sample Module, as shown in Figure 3.
Reagent Stopcock: This lever allows liquid from the syringe to be injected when it is in the horizontal position, and seals off the syringe when it is in the up position.
Clamp Screw: This screw secures the lower half of the Sample Module to the Upper half of the Sample Module when it is tightened. Note there is a pointed and rounded pin in the lower half of the Sample Module. The Clamp Screw clamps against the pointed pin.
Three sizes of Test Tube o-rings are supplied to accomodate variations in sample Test Tube sizes.
Air Module Controls Description
The Air Module provides the air supplies required by the Time Zero Module.
When restarting the Air Module, be sure there isn't a test tube installed on the Sample Module. This prevents the pump from stalling on startup.
There are five controls, three air outputs, and one readout on the front of the Air Module, as shown in Figure 4.
Figure 4
Pinch Air Off/On: When this switch is On, the Pinch Valve on the Sample Module is activated. This prevents saline backflush into the sample.
Pinch Air Output: This air line is connected to the Pinch Valve via the Electronics Module.
Vent Off/On: When this switch is On, the air in the Sample Module is vented to atmosphere. This also switches the cytometer from Ready to Standby. The middle position vents the pump for eaiser restarts.
Pressure Gauge: This indicates the pressure being provided by the Air Module in Pounds per Square Inch. If the output air lines from the Air Module are open, the pressure will read zero. The lines must be connected to the Time Zero Electronics Module for correct readings to be obtained.
Pressure Readout Switch: This switch determines whether the Pressure Readout indicates Run or Boost pressure.
Sample Air Knob: This knob adjusts the Sample Air Pressure. It is adjustable from 0 up to the Boost Pressure. It is usually set to about 6 PSI.
Sample Air Output: This line carries Sample Air to the Time Zero Electronics Module.
Boost Air Knob: This knob adjust the Boost Air Pressure. It is adjustable from 0 up to 35 PSI. It is usually set to about 12-16 PSI.
Boost Air Output: This line carries Boost Air to the Time Zero Electronics Module.
There is one control and one connector on the rear of the Air Module, as shown in Figure 5.
Figure 5
Power Switch: This turns the Air Module on.
Ready/Standby Control Connector: This connector controls the Run/Standby feature. When the Vent Switch is On, the cytometer is switched to Standby, which stops the sheath flow through the cytometer's flow cell. This conserves sheath fluid.
Fuse Holder: The Air Module uses a slo-blow 2A fuse for 117 Volt operation, and a 1A fuse for 220-240 Volt operation.
Running Samples
Figure 6 shows the Sample Module in relation to the FACScan, FACSort, FACSCalibur and LSR. The FACSCalibur configuration without a Loader requires a 5.5" high lab jack placed under the Sample Module to bring it up to the Sample Input Tube. The LSR requires a mounting bracket which mounts the Sample Module to the LSR table.
Figure 6
Leave the Test Tube Lever off to the side if you don't mind having the Droplet Containment pump running while using the Time Zero System.
If you want to disable the Droplet Containment pump while using the Time Zero System, place the Test Tube Lever in the run position. Remove the Sample Tube adjustment nut, and route the Time Zero sample line through the hole where the nut was installed.
Time Mark Switch:
The Time Mark switch can be set to either 0.1 second or 1 second. If the time resolution in the kinetics software is less than 0.25 seconds, the Time Mark switch should be set to 0.1 second. If the time resolution is greater than 0.25 seconds, the Time Mark switch should be set to 1 second.
Boost Time Adjustment:
Next, it is necessary to adjust the boost time. Switch the Vent on the Air Module Off, and the Readout Switch to Boost. Disconnect the two air lines between the Electronics Module and Sample Module. This dead ends the air lines. Adjust the boost air pressure to 15 PSI with the Boost Air Knob. Switch the Readout Switch to Run, and adjust the sample air pressure to 6 PSI with the Sample Air Knob. Reconnect the two air lines. Switch the Pinch Air and Vent On.
Loosen the Clamp Screw, and separate the lower half of the Sample Module from the upper half. Put a tube of beads, such as FITC Calibrite beads, in the lower half of the Sample Module. Reinstall the lower half into the upper half, and tighten the Clamp Screw. Adjust the Boost Time to mid range. Slide the cytometer's Test Tube Lever to the right. If necessary, remove the cytometer's Droplet Containment Tube to connect the Time Zero's Sample Line to the cytometer as shown in Figure 6. This is done by loosening the nut at the top of the stainless steel input line, and gently pulling the Containment Tube down.
Close the Sample Module's Reagent Stopcock (Up). Put the Cytometer in Run. Switch the Vent and Pinch Air Off.
FACScans and FACSorts should switch from Standby to Ready at this point.
On FACSCalibur and LSR, the Run button should be glowing green rather then yellow to indicate the fluidics and optics are in the Run mode rather than the Soft Standby mode.
Adjust the sample flow rate using the Sample Air Knob on the Air Module.
Next, switch the Vent On. This allows the Sample Line to backflush into the tube of beads, and clears beads from the Sample Line.
Then simultaneously press the Boost Button and switch the Vent Off. Listen for the boost valve switching, indicating the end of the boost. If the boost time is properly adjusted, events should appear on the computer screen at the end of the boost. If events begin after the end of boost, increase the boost time; conversley, if events begin before the end of boost, decrease the boost time. To try a different boost time, switch the Vent On for a few seconds to allow the Sample Line to backflush, then simultaneously switch the Vent Off and press the Boost Button. After establishing the boost time (it should be about 0.5 seconds), switch the Vent On, remove the lower half of the Sample Module, and allow the Sample Line to backflush, switch the Pinch Air On, and put the Cytometer in Standby.
To confirm the correct boost time has been set, fill the Reagent Syringe with a type of marker bead that is easily distinguished from the FITC Calibrites. Close the Reagent Stopcock (Up), and install the Reagent Syringe in the upper half of the Sample Module. Switch the cytometer to Run, install the lower half of the Sample Module containing the FITC Calibrite beads, tighten the Clamp Screw, switch the Vent and Pinch Air Off, and establish normal flow of the FITC Calibrites. Open the Reagent Stopcock (Down). Simultaneously press the Boost Button and inject the marker beads in the Reagent Syringe. Generate a display of the results, which would typically be FL1 vs time. A properly adjusted boost time will result in the marker beads appearing at the end of the boost time mark, as shown in Figure 7.
Figure 7
Appearance of the marker beads before the end of the boost time mark indicates the boost time is too long. Conversely, if the marker beads appear after the end of the boost time mark, the boost time is too short. Once the boost time is set, it doesn't need to be reset unless the length or diameter of the sample tubing is changed.
If the boost time is shorter than the length of the time mark, the two time marks will merge into one.
Running Samples:
After the boost time is adjusted, you are ready to run samples. Place cells and the mixing flea into a test tube, and place the test tube in the lower half of the Sample Module. Adjust the mixing speed. If temperature control is desired, turn on the recirculating water bath, and run it until the desired temperature is displayed on the Electronics Module. Set the Cytometer Fluidics to Run, and switch the Vent and Pinch Air On. Install the lower half of the Sample Module into the upper half, and connect the Time Zero Sample Line to the cytometer. Tighten the Clamp Screw. Close the Reagent Stopcock (Up). Load the Reagent Syringe, and install it in the upper half of the Sample Module. Switch the Vent and Pinch Air Off. Adjust the sample flow rate using the Sample Air Knob. Adjust PMTs and gates if necessary. Open the Reagent Stopcock (Down). Stmultaneously inject the Reagent and press the Boost Button. Additional reagent may be added by closing the Reagent Stopcock and reloading the Syringe. After data has been collected, switch the Vent On, close the Reagent Stopcock, loosen the Clamp Screw, and remove the lower half of the Sample Module, and allow the Sample Line to backflush. Switch the Pinch Air On to stop the backflush. Clean the exterior of the Sample Tube with a squeeze bottle containing sheath fluid.
At the end of an experiment, it is a good idea to run distilled water as sample, to prevent saline from crystalizing in the Time Zero Sample Line. Disconnect the Sample Line from cytometer while the distilled water is flowing to prevent the sheath fluid from backflushing into the Sample Line.
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Comment #1 (Posted by ray hicks
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tz user guide
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