Alternative CTC Staining Protocol

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REAGENTS

  • Mouse Gamma Globulin (Jackson Immuno 015-000-002, store sterile at 4°C)
  • Mouse anti CD45-PE, human (clone: 5B1) leukocytes (Miltenyi catalog number 130-098-141)

Optional: We recommend using the above anti CD45 in combination with any or all of the followings:

  • Mouse anti CD66b-PE, human (clone: REA306) granulocytes (Miltenyi catalog number 130-104-414)
  • Mouse anti CD38-PE, human (clone: IB6) white blood cells (Miltenyi catalog number 130-098-907)
  • Mouse anti CD27-PE, human (clone: M-T271) white blood cells (Miltenyi catalog number 130-097-926)
  • Fixative Solution (3.7% formaldehyde solution, protect from light, store at 4°C)
  • TX-100 10% Solution
  • Mouse anti human pan Cytokeratin FITC-conjugated antibody (protect from light, store at 4°C)
  • Hoechst 33342 (protect from light, store at -20 to 4°C)
  • Mounting medium (protect from light, store at -20 to 4°C)
  • Human BD Fc Block™ (BD 564219)

ENUMERATION PROCEDURE

SAMPLE STAINING

Note before starting: This preparation is for one sample; scale up as appropriate. Alternatively, all solutions may be prepared freshly before staining. Be mindful during liquid handling steps to retain the cells/beads pellet (i.e. try not to lose the beads in the pipet tips). Direct pipetting of cells/beads should be minimized and very gently if done. All staining procedures are to be performed at room temperature and protect from light.

  1. After CTC isolation with IsoFlux system, transfer the cells/beads into a clear microfuge tube using Binding Buffer (BB). Place the tube on the large round magnet for 30 seconds, remove and discard the buffer. Remove the tube from the magnet. Immediately dispense 36mL of BB into the tube. Add 4mL of Mouse Gamma Globulin (This is approximately 1mg/mL final concentration) to the tube. Gently flick the tube to disperse the cells/beads, making sure that the sample/beads do not stick to the wall of the tube.
  2. Incubate at room temperature for 5 minutes.
  3. Add 4mL of anti-CD45-PE antibody to the tube (i.e. 1:11 dilution). Incubate in the dark at 4°C for no more than 20 minutes. Gently flick the tube occasionally to mix. If adding the optional anti CD27, CD38, and/or CD66b antibodies, use 1mL of each.
  4. Place the tube on the large round magnet for 30 seconds, remove and discard the antibody buffer. Remove the tube from the magnet. Add 100mL of BB. Gently disperse the cells/beads sample. Place the tube on the magnet for 30 seconds and discard the buffer (This is called washing with magnet pull-down method).
  5. Dilute the Fixative solution in BB at 1:1 ratio. Add 40mL of the diluted Fixative solution to the sample and incubate in the dark at room temperature for 20 minutes.
  6. Place the tube on the large round magnet for 30 seconds, remove and discard the Fixative solution. Wash the sample with 100mL BB using the pull-down method as mentioned above. (Potential stopping point: sample may be stored in the dark at 4°C up to 3 days. However, we recommend completing the staining and acquiring data as soon as possible because PE is easily bleached.)
  7. Make 0.2% TX-100 solution in BB (this is called Permeabilization buffer). Place the tube on the large round magnet for 30 seconds, remove and discard the BB. Add 40mL Permeabilization buffer to the tube and incubate at Room Temperature for 5 minutes.
  8. Place the tube on the large round magnet for 30 seconds, remove and discard the Permeabilization buffer using the pull-down method.
  9. Add 36mL of BB to the sample. Add 4mL of Permeabilization buffer to the sample (this is to make 0.02% TX-100 solution). (Optional: Add 4mL of Fc Blocking reagent and incubate at Room Temperature for 5 minutes.)
  10. Add 4mL of anti CytoKeratin-FITC to the tube and incubate in the dark for 40 and no more than 60 minutes at room temperature. Gently flick the tube occasionally to mix.
  11. Place the tube on the large round magnet for 30 seconds, remove and discard the CK staining solution. Wash the sample with 100mL of 0.02%TX-100 in BB using the pull-down method above. Remove the solution.
  12. Add 100mL of BB containing 1X Hoechst 33342 and incubate at room temperature for 10 seconds.
  13. Transfer the stained cells/beads into a SensoPlate™ (alternatively, glass slide may be used) well centered on a small round magnet. Remove the buffer while keeping the stained cells/beads at the center of the well over the magnet.
  14. Remove the plate from the magnet. Dispense 3mL of Mounting Media over the cells/beads spot. Immediately place the cells/beads over the magnet for a few seconds. Place the glass coverslip over the cells/beads spot. Remove the plate from the magnet. Note that PE signal is better preserved when the mounting media is slightly diluted with BB; pure PBS will quench the PE signal.
  15. Cover the SensoPlate™ with plate sealer and aluminum foil. The sample is now ready for imaging. We recommend that the image acquisition be done as soon as possible to maintain the Phycoerythin (PE) signal.