CELLRAFT™ SYSTEM - INSTRUCTIONS FOR USE
Preparation and Cell Culture
The following steps describe the method to prepare and utilize the CellRaft™ Array for cell culture and
identification.
1. 2. 3. 4. 5. 6. 7. 8. In a sterile tissue culture hood, remove the CellRaft Array from the sealed pouch.
Remove the lid and add 2 mL of PBS or culture medium to the CellRaft Array.
Wait three minutes, then remove the PBS or medium.
Repeat steps 2-3 two additional times. Note: do not allow the CellRaft Array to dry. This will cause air
bubbles to form in the microwells that will prevent cell adhesion and impair imaging.
If necessary, coat the CellRaft Array with desired extracellular matrices as per standard protocol (again
ensuring that the CellRaft Array does not dry). Otherwise proceed to step 6.
Generate a single-cell suspension in 2-3 mL of desired culture medium. It is recommended to use 4,0006,000 cells for 200 µm x 200 µm CellRaft Arrays and 15,000-22,000 cells for 100 µm x 100 µm
CellRaft Arrays. Note: these recommendations maximize the number of single cell captures per CellRaft
while minimizing the number of multi-cell captures.
Add cells in suspension evenly across the entire CellRaft Array. Note: single cells should attach to the
CellRaft Array within 2-6 h, depending on cell type.
Once adhered, screen the CellRaft Array using brightfield, fluorescence, or confocal microscopy to
identify target cells. Use the provided location chart to record the location of cells of interest.
CellRaftTM System Setup
The following describes the setup and calibration of the CellRaft™ System on a standard inverted microscope.
1. 2. 3. 4. 5. 6. 7. 8. Remove the CellRaft Release Device (needle release device, controller and power supply) from the case.
Place the controller on a flat surface near the microscope.
Lower the turret to the lowest possible position and remove any stage inserts present.
Feed the four-color connecting wire from the control box up through the stage from underneath and
connect to the release device.
Place the CellRaft Needle Release Device on the microscope objective.
Holding the tabs on the collar of the Release Device, turn the collar clockwise to tighten the Release
Device around the microscope objective.
Remove the cap from the release device. Note: if the needle contacts any surface other than the CellRaft
Array, it must be replaced.
Plug the power supply into a wall outlet and wait for the green light on the controller to turn on.
Setup Mode and Release Device Calibration
1. Turn the “Travel Distance” knob counterclockwise until it stops.
2. Hold the “Release” button for three seconds. The green light will begin to blink, as the controller has
entered setup mode.
3. Turn on a camera and monitor attached to the microscope (a camera and monitor are suggested for cell
identification and CellRaft release).
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4. While watching the monitor, gradually increase the “Travel Distance” knob by turning clockwise.
Continue until a small, white circle can be seen on the screen. This circle is the release needle.
5. Using the provided Post-It® Arrow Flags, place an arrow on the monitor screen to indicate the location
of the release needle.
6. Press and release the “Release” button to return release mode.
CellRaft Release
The following steps describe the method to collect cells of interest from the CellRaft™ Array.
1. Place the CellRaft Array on the microscope stage. The CellRaft Array can be fixed in place with stage
clips if available, but fixing is not necessary.
2. Focus on the CellRaft Array and ensure the array is in the appropriate orientation to read the numbers
located on the array elements.
3. Move the stage to place a CellRaft of interest under the Post-It® Arrow Flag indicating the release
needle location.
4. Press and release the “Release” button. Examine the CellRaft Array to determine if the desired CellRaft
was released. If the CellRaft was not released, slightly increase the needle travel distance by turning the
“Travel Distance” knob clockwise. Repeat the release process and examine again for CellRaft release.
Repeat this process until the CellRaft has been released. Once the CellRaft is released, the travel
distance should no longer require adjustment.
Collecting Released CellRafts
The following steps describe the various methods used to collect and transfer CellRafts.
Collection and transfer using the Standard Wand.
1. Remove the Standard Wand from the CellRaft™ System case.
2. Sterilize the Standard Wand by spraying with 75% ethanol and allowing to dry (~30 min) in a sterile
tissue culture hood.
3. While the Standard Wand is drying, place the Multiwell Collection Plate on a level surface near the
microscope. Use caution not to loose control of the plate, as plate contains strong magnets and will be
attracted to metal surfaces.
4. If using a 96-well plate, fill desired wells with 200 µL of medium. Other standard plate style reservoirs
(6-well, 12-well, etc.) may be used, but will require various amounts of medium. Add enough medium to
the reservoirs to ensure that the bottom 3 mm of the Standard Wand will be submerged in the medium.
5. Place the CellRaft Array in the sterile tissue culture hood.
6. Remove the lid from the CellRaft Array and place the Standard Wand atop the array so that the disk
attached to the wand covers the CellRaft Array reservoir.
7. Move the CellRaft Array to the microscope stage.
8. Release CellRafts as described above. Once released, CellRafts should be seen migrating toward the
Standard Wand. If the CellRafts to not migrate toward the Standard Wand, move the wand closer to the
release site and attempt the release again. Repeat until the CellRaft migrates toward the wand. Wait 5
seconds to ensure CellRaft is collected.
9. Lift the Standard Wand vertically off of the CellRaft Array.
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10. Move the Standard Wand to hover over the center of the Multiwell Collection Plate. The magnet within
the wand should levitate and is usually accompanied by a clicking sound.
11. Move the Standard Wand to the desired collection well. Lower the wand into the well and gently shake
for 5 seconds.
12. Lift the Standard Wand vertically out of the well.
13. Return the Standard Wand to the CellRaft Array. The magnet should fall and be visible at the base of the
wand.
14. Repeat steps 8-13 to collect additional CellRafts.
Collection and transfer using the MiniWand
1. Remove the MiniWand from the CellRaft™ System case and its containing tube.
2. Sterilize the MiniWand by soaking in 1 mL of 75% ethanol in a 1.5-mL microcentrifuge tube for 10
min.
3. Prime the MiniWand by soaking in a PCR tube containing at least 7 µL of lysis buffer for 10 min.
4. While the MiniWand is drying, place the PCR Collection Plate on a level surface near the microscope.
Use caution not to loose control of the plate, as plate contains strong magnets and will be attracted to
metal surfaces.
5. Rinse the MiniWand by a brief immersion in a 1.5-mL microcentrifuge tube containing 1 mL PBS.
Repeat this step anytime the wand is exposed to lysis buffer. Store the MiniWand in a 1.5-mL
microcentrifuge tube containing 1 mL PBS when not in use.
6. Place the CellRaft Array on the microscope stage.
7. Release the CellRafts as described above. The lid may be kept in place to help prevent contamination
during release. However, it must be removed to collect CellRafts when using the MiniWand.
8. Once released, remove the lid from the CellRaft Array.
9. Lower the tip of the MiniWand into the medium contained on the CellRaft Array.
10. Bring the MiniWand into proximity of the released CellRaft (2-3 mm). The CellRaft should be seen
migrating toward the MiniWand. Wait 5 seconds to ensure the CellRaft is collected on the MiniWand.
11. Lift the MiniWand vertically out of the medium. Replace the lid on the CellRaft Array.
12. Rinse the MiniWand possessing the collected CellRaft by slowly dipping the wand in a 1.5-mL
microcentrifuge tube containing 1 mL PBS buffer or any desired rinsing agent. Avoid contacting the
tube walls.
13. Hold a PCR tube containing 4-7 µL of lysis buffer above the magnet contained in the PCR Collection
Plate.
14. Slowly lower the MiniWand vertically into the lysis buffer. The magnet within the MiniWand should
levitate. Avoid contacting the tube walls.
15. Withdraw the MiniWand from the lysis buffer.
16. Repeat steps 14-15 a total of 3-5 times.
17. Place the MiniWand in the storage solution (1 mL PBS in 1.5-mL microcentrifuge tube). The magnet
should fall and be visible at the bottom of the MiniWand. If not, shake the wand to allow the magnet to
fall.
Collection and transfer using the Disposable Wand
1. Remove a Disposable Wand from the CellRaft™ System case and its containing tube.
2. Decontaminate the Disposable Wand by soaking in 1 mL of a 5% bleach solution in a 1.5-mL
microcentrifuge tube for 5 min. This procedure may also be performed on the MiniWands prior to use.
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3. Sterilize the Disposable Wand by soaking in 1 mL of 75% ethanol in a 1.5-mL microcentrifuge tube for
10 min.
4. Rinse the Disposable Wand of ethanol by dipping in 1 mL PBS in a 1.5-mL microcentrifuge tube
5. Repeat step 4 serially two times in fresh PBS.
6. Place the CellRaft Array on the microscope stage.
7. Release the CellRafts as described above. The lid may be kept in place to help prevent contamination
during release. However, it must be removed to collect CellRafts when using the Disposable Wand.
8. Remove the lid from the CellRaft Array.
9. Bring the magnet-possessing end of the Disposable Wand into proximity of the released CellRaft. The
CellRaft should be seen migrating toward the Disposable Wand. Wait 5 seconds to ensure the CellRaft
is collected to the Disposable Wand.
10. Lift the Disposable Wand vertically out of the medium. Replace the lid on the CellRaft Array.
11. Rinse the Disposable Wand possessing the collected CellRaft by slowly dipping the wand in a 1.5-mL
microcentrifuge tube containing 1 mL PBS buffer or any desired rinsing agent. Avoid contacting the
tube walls.
12. Place the Disposable Wand into a PCR tube containing 4-7 µL of lysis buffer. The Disposable Wand
may remain in the lysis buffer for as long as desired. The tubing of the disposable wand may also be cut
to any desired length in order to fit the wand in the tube with the tube cap in place if desired.
13. After lysis is complete, remove the Disposable Wand from the tube and discard.
Release Needle Replacement
The following steps describe the process of replacing the release needle.
1. Remove the release device from the microscope object and disconnect from the controller. Note: ensure
the controller is disconnected from the wall outlet prior to disconnecting the release device.
2. Place the release device on a flat surface.
3. Using the provided screwdriver, loosen but do not remove the four set screws found surrounding the
release needle.
4. Invert the release device above the opening of a sharps waste container to dispose of the damaged
release needle and window.
5. Use tweezers to carefully remove the tape holding a replacement release needle in its containing dish.
Handle by the edges of the clear window. Use caution to avoid contacting the needle!
6. Place the replacement needle on the release device.
7. Tighten the four set screws, taking care to ensure that the window is centered on the release device and
sits level. Do not over tighten screws, as they will strip or break and require replacement of the entire
release device!
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