Sample Mounting Kit for IsoFlux CTC Samples

MagRack, MagStand for staining and mounting sample onto plate. Use with kit catalog No. 910-0124

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INTENDED USE

This kit includes accessories designed to increase both repeatability and ease of use for the staining and fixation of IsoFlux samples intended for microscopic enumeration. This kit is included as a standard component for IsoFlux systems sold on or after January 2019, and can be purchased for existing systems in the field.

SUMMARY AND EXPLANATION

Previous instructions were to use either glass bottom 24-well plates (e.g. SensoPlate 24well from Greiner) or glass slides in combination with magnets provided in order to stain and mount IsoFlux samples for imaging as described in the IFU for the CTC enumeration kit (630-0138). The current kit is provided as a replacement for the loose magnets previously provided for processing.

One key component, the MagRack, is a specially designed low volume magnetic rack that enables hands-free staining of IsoFlux samples. This enables processing of up to 8 samples at the same time.

A second component is the MagStand which works to control the dispersion of cells onto the glass bottom plate, while minimizing loss. The result is a bead / cell dispersion area that is centered within each well and provides optimal imaging parameters via either the IsoFlux Imager. IsoFlux glass bottom plates are also compatible with standard inverted microscopes.

PRINCIPLES OF THE PROCEDURE

COMPONENTS

  • IsoFlux glass bottom enumeration plates, 2x 24-well (910-0123) CTC Sample Mounting Accessories:
    • 1x MagRack (fits 8x 0.5mL microfuge tubes) o 1x MagStand (for use with glass-bottom 24-well plate) o 1x MagPaddle 2.5 (with 0.25in diameter magnets) o 1x MagPaddle 5.0 (with 0.50in diameter magnets, red)
      MATERIALS REQUIRED, NOT PROVIDED 
  • 12mm diameter round glass cover slips
  • tweezers, non-magnetizable
  • mounting media (included in the IsoFlux enumeration kit)

WARNINGS AND PRECAUTIONS

           Please use protocols compatible with the handling of blood specimens.

Staining Procedure

  1. Use 0.5mL protein LoBind tube and the MagRack while following the
    Enumeration Kit instructions (630-0138)
  2. Whenever placing tubes on the magnet is required, place the tube in the rack and wait 5 seconds or until the beads completely accumulate to the side of the tube.
  3. Remove the buffer with micropipette tip without disturbing the beads pellet.
     
  4. Upon completion of the staining procedure add 100L of BB to the cells/beads. Set aside.

Mounting Procedure:

  1. Insert the MagPaddle2.5 to the MagStand corresponding to the desired wells, working from top to bottom and left to right respectively (i.e. for a new plate, start with position A1, B1, C1, D1)
  2. Place the glass bottom plate onto the MagStand with MagPaddle2.5 in place.
  3. Transfer the stained cells/beads into the corresponding wells at the magnet center.
  4. Remove excess fluid by aspirating in the area outside of the magnet. Rinse tubes with the excess fluid and add back to the well until no beads is left in the tube, avoid forming bubbles.
  5. Remove excess fluid from the well and discard. Aspirate off the excess fluid to dry as much as possible such that the sample appear wet but no dome of fluid on top of sample should remain.
  6. Continue with other samples for the remaining wells. Two samples are shown in this example.
  7. Remove MagPaddle2.5 from the MagStand by lifting it in a straight and upward direction. Avoid sliding the plate as it may disturb the sample.
  8. Dispense 2-3 µL of Mounting Media over the center of cells/beads spots. This helps disperse the beads so that it will be evenly spread out, allowing for enumeration
  9. Insert MagPaddle 5.0 to the same column.
  10. Place the glass bottom plate (with samples) onto the MagStand.
  11. Place the glass 12mm cover slips over the cells/beads spots. Remove the plate from the magnet by lifting it in a straight and upward direction.
  12. Samples are now well dispersed and ready for the next step.
  13. Cover with plate sealer and aluminum foil. The sample is now ready for imaging and can be stored at 4oC for up to 2 weeks if kept moist and away from light.

2.14     Below is a stitched image of an entire well with beads and the cover slip applied: