Instructions for Use: Rare Cell Enrichment Kit - Streptavidin

Applies to: 910-0103

Updated: 2023-04-07
Revision: D

INTENDED USE

The intended use for the IsoFlux™ Rare Cell Enrichment Kit (Streptavidin) (RCE Kit) is as a general-purpose laboratory reagent for the enrichment of rare cells in the blood circulation. The kit is used with the IsoFlux System, a benchtop instrument for semi-automated cell isolation. The kit contains an immunomagnetic bead reagent functionalized with streptavidin. It will bind biotin-labeled antibodies and other similar molecules (all referred to as biotinylated antibodies in the following text). The RCE kit is for Research Use Only.

SUMMARY AND EXPLANATION

Many rare cell types of scientific and clinical interest exist in the peripheral blood circulation. These include circulating tumor cells (CTCs), cancer stem cells, immune cells, and circulating endothelial cells. In many cases, these cells can be preferentially enriched from the background cells using differentially expressed cell-surface antigens.

The IsoFlux RCE Kit is designed to standardize and automate the enrichment of rare cells from biological samples using the IsoFlux System. Cells are positively isolated from the sample using an immunomagnetic capture reagent while the sample flows through a microfluidic cartridge designed for cell isolation. The kit produces an enriched cell pellet that can subsequently be used for further testing.

PRINCIPLES OF THE PROCEDURE

The IsoFlux RCE Kit contains streptavidin-coated capture beads (RCE beads), microfluidic cartridges, and additional reagents required for performing rare cell enrichments. The RCE beads consist of micro-scale particles with a magnetic core surrounded by a polymeric layer coated with streptavidin. RCE beads in combination with biotinylated primary antibodies (selected by the user, based on specific application) are ideal for enrichment of rare cells, depending on the specificity of the primary antibodies or similar molecules. Rare cells can be isolated from mononuclear cell suspensions of whole blood or other similar cell samples. The primary antibodies are either pre-coated onto the RCE beads (direct method) or added to the cell sample (indirect method). RCE beads are then mixed with the cell sample to bind to the target cells during a period of incubation.

The cell sample and beads mixture is loaded onto the microfluidic cartridge and processed with the IsoFlux instrument, where the cells pass through the fluidic channel of the cartridge. Midway through the fluidic channel is a cell isolation zone that is exposed to an external magnetic field inside the instrument. The target cells, having RCE beads attached, are attracted towards the magnetic field. The target cells are collected on a removable disk that forms the roof of the isolation zone. After the sample is processed, the enriched cells are transferred inside the instrument to low volume recovery holder or a microfuge tube. The enriched rare cells are ready for further analysis.

MATERIALS PROVIDED

• Instructions for Use
• 8 sterile microfluidic cartridges (includes 8 low-volume recovery holders, 8 microfuge tubes for cell recovery)
• 500µL RCE (streptavidin-coated) beads*
• 500µL Fc blocker reagent*
• 5 x 12mL sterile preservative free Binding Buffer
  *Contains 0.02% sodium azide as a preservative.

REAGENT STORAGE AND HANDLING

• Microfluidic cartridges should be stored unopened at room temperature.
• RCE beads and Fc blocker reagents should be stored at 2° to 8°C and used within 60 days after opening.
• Binding Buffer should be stored unopened at 2° to 8°C. After opening. Unused buffer may be stored frozen at -20°C, thawed and used once within 60 days.
• Protect reagents from heat in excess of 35°C. Do not freeze.
• 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.

MATERIALS REQUIRED, NOT PROVIDED

• IsoFlux Instrument (Catalog No. 950-0100)
• Swing bucket centrifuge capable of 1500xg (with brake settings)
• Test tube racks
• Calibrated micro-pipettors and tips
• Serological pipettes and pipettor
• 1.5 or 2mL microfuge tubes (preferably low retention)
• Microfuge tube rotator
• 50mL Leucosep® tubes (with frit) (Greiner, Catalog No. 227290)
• Phosphate Buffer Saline without Catt Mg** (PBS-CMF)
• 50mL conical tubes
• Ficoll-Paque™ Plus (GE Healthcare, Catalog No. 17-1440-02)
• Permanent magnets (accessory parts included with IsoFlux instrument, large round and small cylindrical magnet)
• CTL-Wash™ Supplement (CTL, Catalog No. CTLW-010)
• Optional: Benzonase® Nuclease (Sigma, Catalog No. E8263)
• Optional: Nylon Mesh Cell Strainer, 40 Micron (BD, Catalog No. 352340)
• Optional: RPMI tissue culture media

WARNINGS AND PRECAUTIONS

• For Research Use Only
• Please read the entire contents of the Instructions for Use before processing samples.
• Caution: Care should be taken to collect and transfer blood samples before processing. Cells are fragile and can be damaged or lost if not handled properly.
• 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.

ENRICHMENT PROCEDURE

Specimen collection and preparation

1. Collect biological samples aseptically into an appropriate sample collection tube.
2. If samples are being shipped or transported, pack the samples accordingly to control exposure to excessive temperatures or agitation. Typically, samples can be shipped in an insulated Styrofoam shipping container with cold (not frozen) gel packs as a buffer to temperature fluctuations.
3. Depending on the sample type, a pre-processing step might be required such as density centrifugation or red blood cell lysis.
4. A typical pre-processing procedure for human whole blood (mononuclear cell fraction preparation) is provided in Section 2. Please consult the manufacturer's instructions for pre-processing procedure for other cell sample types.

Pre-processing of whole blood sample (mononuclear cell fraction preparation)

1. A 7 to 10mL peripheral blood sample should be drawn into anti-coagulant K2EDTA coated blood collection tube and kept at room temperature. Whole blood sample should be processed within 36 hours of collection.
2. For each sample to be processed, coat a 2mL tube with 1mL of Binding Buffer (or 0.6mL if using 1.5mL tube) and rotate at 4°C until use.
3. Prepare the 50mL Leucosep (with frit) tube by adding 15mL of Ficoll-Paque PLUS and centrifuging the tube at 1000xg for 30 seconds with the brake setting to ON.
4. Only when ready to process the blood sample, gently add 5mL of PBS-CMF to the Leucosep tube.
5. Immediately add blood sample to Leucosep tube by first decanting it from the blood collection tube into the Leucosep tube. Then, gently wash down the walls of the blood collection tube twice, each time with 10mL of PBS-CMF.
6. Immediately centrifuge the tubes at 800xg for 15 minutes with the brake setting to OFF.
7. Decant about 20mL of the supernatant in Leucosep tube into a new 50mL conical tube. Gently swirl the remaining supernatant to dislodge any cells that may be stuck to the wall of the Leucosep tube and then decant it into the same conical tube. Rinse the wall of the Leucosep tube with 5mL of PBS- CMF and add that to the same 50mL conical tube. Be careful not to suction the Ficoll-Paque™ PLUS through the frit; avoid pressing the pipette against the frit. Optional: CTL-Wash™ Supplement may be added to improve cell viability.
8. Centrifuge at 280xg for 10 minutes with the brake setting to ON.
9. Use a 25 or 50mL serological pipette to gently aspirate off the supernatant as much as possible without disturbing the pellet. Use a 5mL pipette to remove the remaining supernatant closer to the bottom of the tube and avoid disturbing the pellet (up to ~500µl buffer may be left remaining). Keep the pellet on ice. We recommend that you do not decant the supernatant, because the pellets might be very loose in clinical samples.
10. Gently tap the tube on the bench a few times to loosen pellet. Tap patiently until the pellet is completely resuspended. If necessary, add up to 300µL of Binding Buffer to the tube. All cell clumps must be dispersed as they may clog the micro-channel during isolation. Gently resuspend the cells with P1000 pipette.
11. Wash PBMC with 30mL of PBS-CMF with CTL wash (or RPM media). Centrifuge at 280xg for 10 minutes. Discard supernatant.
12. Resuspend PBMC in 0.7mL of BB. Add 0.1 mL CTL wash supplement. Optional: Benzonase Nuclease (up to 1000 Units per sample) may be added to minimize cell aggregation due to cell lysis. *Benzonase Nuclease should be avoided if performing nuclease sensitive down-stream analysis.
13. Remove and discard the Binding Buffer from the tube prepared in Step 2.
14. Transfer cell suspension into the 2mL tube. Rinse the residual cells in the 50mL conical tube with Binding Buffer and transfer to the same 2mL tube. The final volume should be approximately 1mL. Keep cell suspension on ice until use.

Coupling reaction of beads and cell sample:

Coupling method (direct or indirect) selection: Depending on antibody and target cells, direct or indirect method may yield better results. If a cocktail of many primary mouse antibodies is used, the indirect method is recommended. Users should titrate the optimal amount of beads to be used per sample. As the starting point, 12.5µL beads suspension may be used for 1mL of cell sample (e.g. mononuclear cells preparation from 7-10mL of whole blood or ~2 x 10' cells). As an example, a bead titration study of 12.5, 25 and 50µL beads may be done to access target recovery and background cells to decide the optimal amount of beads to use.

Indirect method (add biotin-labeled antibody to cell sample)

Use approximately 10µg of biotinylated primary antibody (for multiple capture antibodies, use approximately 10µg of each primary antibody) per 1mL of cell sample (e.g. mononuclear cells preparation from 7-10mL of whole blood or ~2 X 10' cells). Titrate the primary antibody to optimize the amount used.

1. Add 40µL of Fc Blocking Reagent to the 1mL cell sample (prepared in Section 2). Mix gently by inverting the tubes a few times and incubate on ice for 5 minutes.
2. Add the biotinylated antibody to the cell sample. Incubate for ≥30 minutes at
   4°C with gentle tilting and rotation.
3. Transfer the sample to a 15mL a conical tube. Wash the cells with 5mL PBS-

   CMF with CT wash (or RPM media) and centrifuge at 280xg for 10 minutes.
   Discard the supernatant.

4. Resuspend the cells in 0.9mL of BB. Add 0.1mL CTL wash supplement.
5. Resuspend the RCE beads stock with a micropipette. Dispense 120µL of beads stock into a microfuge tube containing 1mL of Binding Buffer. Place the tube on the large magnet for 1 minute (or until the supernatant is clear) and discard the supernatant. Remove the tube from the magnet and resuspend the washed beads in 120µL Binding Buffer (This preparation suffices for 8 samples. Scale up or down as appropriate).
6. Add 12.5µL washed RCE beads to the cell suspension.
7. Incubate for 2 hours at 4°C with gentle tilting and rotation. The cell sample is ready for rare cell enrichment by IsoFlux System.

Direct method (coat RCE beads with biotinylated primary antibody)

This preparation suffices for 8 samples. Scale up or down as appropriate.

1. Use 2-6µg of biotinylated primary antibody per 120µL of washed beads. It is recommended to titrate the biotin-labeled primary antibody to optimize the amount used.
2. Resuspend the RCE beads stock with a micropipette. Dispense 120µL of a beads stock into a microfuge tube containing 1 mL of Binding Buffer. Place the tube on the large magnet for 1 minute and discard the supernatant. Remove the tube from the magnet and resuspend the washed beads in 120uL Binding Buffer.
3. Add the biotinylated primary antibody to the 2mL microfuge tube containing 120µL pre-washed beads.
4. Incubate for ≥1 hour at 4°C with gentle tilting and rotation.
5. Place the tube on the large magnet for 1 minute (or until the supernatant is clear) and discard the supernatant.
6. Remove the tube from the magnet and wash the beads twice with 1mL of
   Binding Buffer.
7. Place the tube on the large magnet for 1 minute (or until the supernatant is clear) and discard the supernatant. Resuspend the beads in 120µL of Binding Buffer. Keep on ice until use.
8. Add 40µL of Fc Blocking Reagent to the cell sample (prepared in Section 2). Mix gently by inverting the tubes a few times and incubate on ice for 5 minutes.
9. Add 12.5µL of biotinylated antibody coated beads (Step 7) to the cell sample. Incubate for 2 hours at 4°C with gentle tilting and rotation. The cell sample is ready for rare cell enrichment by IsoFlux System.

CTC enrichment with IsoFlux System

1. Refer to the IsoFlux System Instructions for Use and on-screen commands for full instructions to process samples for cell enrichment.
2. Power on the IsoFlux instrument. The touch screen panel will light up; the instrument will initialize and perform automatic routine system check.
3. The touch screen will display “Run Protocol” and “Select Protocol” icons when the instrument is ready for use. Choose one the options below:
   1. “Run Protocol” to run the most recent protocol used (shown at the bottom of the touch screen).
   2. “Select Protocol” to select the appropriate protocol and then press “Run Protocol”.
4. Select the Number of Samples to Run. Cartridge loading carriage(s) will slide out automatically. A total 4 samples can be processed simultaneously. Positions No. 1 and 2 are on the left carriage. Positions No. 3 and 4 are on the right carriage. Samples should be loaded from left to right sequentially from Position No. 1 to 4.
5. Remove the microfluidic cartridge from the pouch and position it upright on a flat surface (see drawing below).
6. Sample retrieval  Microfuge Tube is in well No. 5.
7. The Low-Volume Recovery Holder is in well No. 3.
8. Remove and keep the Low-Volume Recovery Holder to be used in the next step.
9. Decide if the enriched cells will be recovered with Low-Volume Recovery Holder or in the Microfuge Tube.
10. If the enriched cells will be recovered with Low-Volume Recovery Holder, remove the Microfuge Tube in well No. 5 and insert the Low Volume Recovery Holder as shown below:
    
11. If using the Microfuge tube, add 300µL of Binding Buffer to the Microfuge tube in the sample retrieval position on the cartridge (well No. 5) as showing below.
    
12. Carefully open the cartridge lid and add 3mL Binding Buffer to the buffer reservoir (well No. 2) of each cartridge. Carefully snap close the cartridge lid.
13. Load cartridge(s) onto the carriage(s). Press Prime. Cartridge loading carriage(s) will slide in automatically. Machine will prime for about 6 minutes.
14. After priming is completed, touch screen will show Ready to Load Sample. Press Ready to Load Sample. Left carriage will slide out automatically.
    Carefully open cartridge lids.
15. Gently add the beads-coupled cell samples from Step 4 of the Coupling reaction of beads and cell sample section to the Sample well (well No. 1) and avoid forming bubbles. Carefully snap close the cartridge lid and load onto the carriage.
16. After all cell samples are loaded for the left carriage, press Load. Left carriage will slide in automatically. If running one or two samples, cell isolation will start at this point. If running more than two samples, right carriage will slide out automatically. Load the rest of samples and press Load, right carriage will slide in automatically. Cell isolation will start.
17. Cell isolation typically takes about 45 minutes but it may vary for different samples.
18. After cell isolation is completed, touch screen will show Extract Sample. Press Extract Sample. Carriage(s) will slide out automatically.
19. The instrument will place the CellSpot into either the Low-Volume Recovery Holder or the Microfuge Tube.

Cell retrieval (if using Low-Volume Recovery Holder)

1. Remove and invert the holder such that the enriched cells are facing up.
2. Immediately add 20µL (a drop) of Binding Buffer to the CellSpot to prevent cells from drying. 
3. If necessary, place the CellSpot over the small cylindrical magnet for 5 seconds to center the cells/beads pellet. Remove the CellSpot from the magnet.
4. Rinse the micropipette tip with Binding buffer to minimize cell sticking to the tip. Gently aspirate the cells/beads into the pipette tip. Dispense the collected cells/beads into a new microfuge tube (not provided). We recommend doing the liquid-to-liquid transfer (i.e. the microfuge tube should also contain a small volume of Binding Buffer).
5. Place the microfuge tube on the large magnet.
6. Aspirate most of the supernatant and rinse the CellSpot to collect any residual cells/beads. Repeat the previous steps 4-6 until no visible cells/beads are observed on the CellSpot.
   
   
   

Cell retrieval (if using Microfuge Tube)

1. Gently invert the tube 2-3 times until all the cells/beads are suspended in the Binding Buffer at the bottom of the tube.
2. Centrifuge the microfuge tubes briefly to collect all cells. Enriched CTCs are now ready for further testing.