IsoFlux CTC Enumeration Kit

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INTENDED USE

The intended use for the IsoFlux™ Circulating Tumor Cell Enumeration Kit (CTC Enumeration Kit) is as a laboratory reagent for the fluorescence staining of cells to identify circulating tumor cells (CTCs) that are cytokeratin (CK) positive, CD45 negative, and nucleated. The kit is designed for use with the IsoFlux System, a benchtop instrument for semi-automated rare cell isolation. The kit contains antibodies and reagents for immunofluorescence staining. The CTC Enumeration Kit is for Research Use Only.

SUMMARY AND EXPLANATION

Circulating tumor cells (CTCs) are cancer cells that shed from a primary or metastatic tumor and enter the peripheral circulation. Carcinomas are cancers of epithelial origin and include breast, prostate, lung, and colorectal cancers. This document is intended to guide the user through a typical procedure of immunofluorescence staining, fluorescent microscope imaging, and image analysis to enumerate circulating tumor cells (CTCs) of epithelial origin isolated using the IsoFlux instrument.

PRINCIPLES OF THE PROCEDURE

Upon enrichment with IsoFlux system, CTCs are fixed and stained with the fluorescent reagents. The fluorescent reagents include the following: anti-CK-fluorescein isothiocyanate (FITC) is specific for the intracellular protein cytokeratin (characteristic of epithelial cells), anti-CD45-Phycoerythrin (PE) is specific for leukocytes, and Hoechst 33342 stains the cell nucleus. Fluorescent images of stained samples are acquired with microscope equipped with an automatic stage. Images are processed by imaging software and CTCs are enumerated as morphologically intact CK+/CD45-/nucleated cells.

KIT COMPONENTS

  • Mouse Gamma Globulin (JacksonImmuno 015-000-002, store sterile at 4°C)
  • Mouse anti human CD45-PE, human clone 5B1 (Miltenyi 130-098-141)
  • Fixative Solution (protect from light, store at 4°C)
  • Permeabilization Solution (protect from light, store at 4°C)
  • Mouse anti human pan Cytokeratin FITC-conjugated antibody (protect from light, store at 4°C)
  • Hoechst 33342 (protect from light, see preparation below, store at -20 to 4°C)
  • Mounting medium (protect from light, store at -20 to 4°C)

REAGENT STORAGE AND HANDLING

  • Protect reagents from exposure to light.
  • When properly stored, reagents are stable until the expiration date printed on the reagent container or kit box. Do not use expired reagents.
  • Do not mix and match reagents from different kits.

WARNINGS AND PRECAUTIONS

  • For Research Use Only
  • Please read the entire contents of the Instructions for Use before processing samples.
  • Caution: All personnel should follow universal precautions for biological sample handling and use personal protective equipment (i.e., safety glasses, laboratory coat, gloves, etc.).
  • Caution: Microbial contamination of reagents can cause erroneous results and should be avoided.
  • Warning: All biological specimens, cartridges and other materials coming into contact with the specimen(s) are considered bio-hazardous. Handle as if capable of transmitting infection. Treat and dispose of waste using proper precautions and in accordance with local, state, and federal regulations. Never pipette by mouth.
  • Warning: Some of the reagents contain sodium azide as a preservative. If swallowed, seek medical advice immediately. Keep out of reach of children. Keep away from food and drink. Wear suitable protective clothing. Contact with acids liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.
  • Operator training is required to perform the test procedure.       

REAGENTS PREPARATION PRIOR TO USE

  • Hoechst 33342: Dissolve the powder in distilled water to make 20mg/mL stock (5000X final concentration). Further dilute a small quantity with Mounting Medium to make 50X concentration. Store in the dark at -20°C to 4°C. Warm to room temperature before use. Prepare fresh 1X Hoechst in Binding Buffer for each use.

MATERIALS REQUIRED, NOT PROVIDED

  • IsoFlux Instrument (catalog no. 950-0100)
  • Binding Buffer (BB) (Supplied with IsoFlux CTC Enrichment Kit catalog no. 9100091)
  • Fluorescence microscope equipped with automatic stage, excitation/emission filters for FITC (495nm/521nm), PE (565nm/578nm) and Hoechst 33342 (361nm/497nm)
  • Imaging analysis software (MetaMorph by Molecular Devices or similar software)
  • Pipettes and tips
  • Microfuge tubes (clear)
  • Permanent magnets (accessory parts included with IsoFlux instrument, large round and small cylindrical magnets)
  • Optional: 24-well Glass Bottom Microplate (e.g. SensoPlate™ Greiner Bio-One catalog no. 662892) or glass slides
  • Glass coverslips

ENUMERATION PROCEDURE

SAMPLE STAINING

Note before starting: During liquid handling steps, be mindful of retaining cells/beads pellet (i.e. be sure not to lose beads in pipet tips). Direct pipetting of cells/beads should be minimized and be very gentle. All staining procedures are to be performed at room temperature and protect from light.

  1. After CTC isolation with IsoFlux system, using 50L BB, recover and transfer the cells/beads into a clear microfuge tube. Place the tube on the large round magnet for 30 seconds, discard the buffer. Remove the tube from the magnet. Immediately dispense 36L of BB into the tube. Add 4L of Mouse Gamma Globulin (This is approximately 1mg/mL final concentration) to the tube. Gently flick the tube to disperse the cells/beads.
  2. Incubate at room temperature for at least 5 minutes.
  3. Add 4µL 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.
  4. Place the tube on the large round magnet for 30 seconds and discard the buffer. Remove the tube from the magnet. Add 100µL 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. Add 40µL of Fixative solution that had been diluted in BB at 1:1 ratio. Incubate in the dark at room temperature for 20 minutes.
  6. Place the tube on the large round magnet for 30 seconds and discard the Fixative solution. Wash the sample with 100µL BB using the pull-down method above. Remove the buffer.
  7. Add 40µL Permeabilization solution to the tube. Add 4µL anti CytoKeratin-FITC to the tube (i.e. 1:11 dilution) and incubate in the dark for no more than 30 minutes at room temperature. 
  8. Place the tube on the large round magnet for 30 seconds and discard the CK staining solution. Wash the sample with 200µL of Permeabilization solution using the pull-down method above. Remove the solution.
  9. Add 100µL of BB containing 1X Hoechst 33342 and incubate at room temperature for 1 minute.
  10. 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.
  11. Remove the plate from the magnet. Dispense 5L 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.
  12. Cover the SensoPlate™ with plate sealer and aluminum foil. The sample is now ready for imaging. It is recommended that the image acquisition is done as soon as possible to maintain the Phycoerythin (PE) signal.

IMAGE ACQUISITION

A fluorescence microscope equipped with automatic stage and excitation/emission filters FITC (495 nm/521 nm), PE (565 nm/578 nm) and Hoechst 33342 (361 nm/497 nm) is needed for cell imaging and CTC enumeration.

  1. Images are acquired using automatic stage with a stage list covering the whole area of the cells/beads sample spot. Typically, magnifying power of 100X (e.g., 10X eyepiece and 10X objective) is needed for reliable CTC enumeration. Increased magnifying power of 400X (e.g. 10X eyepiece and 40X objective) is necessary to acquire a more resolved image of individual CTCs.
  2. Acquire images of the stained sample under 100X magnification for all three fluorescent channels: CK-FITC, CD45-PE, and Nucleus-Hoechst 33342.

IMAGE ANALYSIS

Imaging analysis software (MetaMorph by Molecular Devices or similar) is required for image analysis for CTC enumeration.

Image analysis and CTC enumeration with MetaMorph software is performed as follows:

  1. Open the acquired 100X magnification images as separate stacks for each of the three fluorescent channels.
  2. Adjust the fluorescent intensity and threshold appropriately for all raw images of the three fluorescent channels to remove the auto-fluorescent signals from the beads (see Appendix for representative images).
  3. Overlay the three stacks of images of the three fluorescent channels.
  4. Enumerate CTCs as morphologically intact CK+/CD45-/nucleated cells.
  5. Total recovered cells (CTCs and background leukocytes) are enumerated as nucleated cell (Hoechst 33342+) counts using MetaMorph integrated morphology analysis. 
  6. Representative images of CTCs may be acquired at higher magnifying power (e.g. 400X magnification).

APPENDIX

Typical CTC images (raw and adjusted)

100X magnification: