CAR (G4S Linker) Cell Enrichment Kit #48669

Cell Enrichment Kit Protocol

NOTE: Kit capacity: ~2 x 10⁹ total input cells

A. Solutions and Reagents

Supplied Reagents

• Releasable Streptavidin Beads (1 bottle, 5 mL #27119)
• Lyophilized DNase I (3 vials, > 15,000 units each #91019)
• DNase I Reconstitution Buffer (1 bottle, 2 mL #47776)
• Biotinylated Rabbit mAb (specific to kit)

Additional Reagents (Not Supplied)

NOTE: Refer to Table 1 for recommended tube sizes and volumes.

• Magnet appropriate for tube size
• Mixer allowing tilting and rotation of tubes
• Phosphate Buffered Saline with BSA (PBS-B): Ca²⁺ and Mg²⁺ free PBS supplemented with 0.1% bovine serum albumin (BSA), pH 7.4
• Phosphate Buffered Saline with BSA and EDTA (PBS-BE): Ca²⁺ and Mg²⁺ free PBS with 0.1% BSA (or 0.1% human serum albumin or fetal calf serum) and 2 mM EDTA (or 0.6% sodium citrate)
• Cell Elution Buffer: RPMI 1640 medium with 1% fetal calf serum (FCS), 1 mM CaCl₂ and 5 mM MgCl₂, pH 7.0–7.4

General Considerations:

• Protocol volumes are suitable for enrichment of 1 x 10⁷ total input cells but are directly scalable up to 2 x 10⁸ cells. For higher or lower total cell input count, refer to Table 1.
• When enriching for cells expressing targets with high variability of expression, titration of biotinylated mAb and releasable streptavidin beads is recommended to optimize purity and yield.
• Keep all buffers cold unless otherwise specified.
• Avoid air bubbles (foaming) during pipetting.

B. Reagent Preparation

NOTE: For optimal DNase I activity, ensure that the Cell Elution Buffer pH is 7.0-7.4.

NOTE: Reconstitute vials of enzyme as needed.

1. Wash Releasable Streptavidin Beads (#27119):
   1. Resuspend stock vial of beads via vortexing >30 sec or tilt and rotate for 5 min.
   2. Transfer the recommended volume of beads for the number of purifications being performed to a tube (refer to step 5 in Table 1).
   3. Add the same volume, or at least 1 mL, of PBS-B and mix.
   4. Place the tube in a magnet for 1 min, discard the supernatant.
   5. Remove the tube from the magnet and resuspend washed beads in the same volume of PBS-B as the initial volume of beads.
2. Reconstitute DNase I:
   1. To each vial of Lyophilized DNase I #91019 (as needed), add 300 μL of DNase I Reconstitution Buffer #47776.
   2. Dissolve the enzyme gently. Vigorous mixing (e.g., pipetting/vortexing too hard) will destroy its activity.
   3. Aliquot reconstituted DNase I into suitable portions.
   4. Store at –20°C.

      NOTE: Thaw immediately before use and keep on ice once thawed. Thawed reconstituted DNase I can be refrozen once.

3. Warm appropriate volume of Cell Elution Buffer for the number of purifications being performed to 37°C (refer to step 13 in Table 1).

C. Cell Preparation

NOTE: Blood and serum may contain soluble factors (e.g., antibodies or cell surface antigens) which can interfere with the cell isolation protocol. Washing the cells once may reduce this interference.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 180-350 x g for 5-10 min will be sufficient to pellet the cells.

1. Collect cells of interest and pellet by centrifugation.
2. Resuspend cells at 1 x 10⁷ cells/mL in PBS-BE at 2-8°C.

D. Cell Enrichment

NOTE: For steps requiring incubations with gentle tilting and rotation, do not perform end-over-end mixing if the volume is small relative to the tube size. Tilt and rotate so the cells and beads are kept in the bottom of the tube.

NOTE: If desired, a small sample of resuspended cells from step 4 can be stained with a fluorochrome-conjugated secondary antibody (e.g., Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414) to determine the percentage of target expressing cells in the input prior to enrichment.

NOTE: Purity of elution fraction can be assessed by flow cytometry using a fluorochrome-conjugated secondary antibody (e.g., Anti-rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414).

1. Add 12.5 µL biotinylated antibody to 1 mL cell suspension, mix gently.
2. Incubate for 10 min at 2-8°C.
3. Wash cells by adding 2 mL PBS-BE, then pellet by centrifugation. Discard the supernatant.
4. Resuspend cells at 1 x 10⁷ cells/mL in PBS-BE.
5. Resuspend pre-washed beads and add 25 µL to cells.
6. Incubate 20 min at 2-8°C with gentle tilting and rotation.
7. Optional: Add 1 mL PBS-BE to limit trapping of unbound cells.
8. Place the tube in a magnet for 2 min.
9. While the tube is still in the magnet, remove and discard the supernatant.
10. Remove the tube from the magnet and add 1 mL PBS-B, pipet 2-3 times or vortex 2-3 sec, replace tube in magnet for 2 min.
11. Repeat steps 9 and 10 at least twice to wash the cells. This step is critical to obtain high purity of CAR positive cells.
12. While the tube is still in the magnet, remove and discard the supernatant.
13. Resuspend bead-bound cells in 400 µL pre-warmed (37°C) Cell Elution Buffer.
14. Add 4 µL reconstituted DNase I.
15. Incubate for 15 min at room temperature with gentle tilting and rotation.

    NOTE: Before transferring released cells to new tube in step 17, pre-coat collection tubes with Cell Elution Buffer for at least 5 min.

16. Pipet thoroughly at least 5-10 times to maximize cell release (avoid foaming).
17. Place in magnet for 2 min, then transfer the supernatant with released cells into a tube pre-coated with Cell Elution Buffer.
18. Remove tube from magnet, resuspend bead fraction in 400 µL Cell Elution Buffer, repeat steps 16 and 17 once to collect residual cells.

Table 1. Recommended Volumes for Different Cell Numbers

*If total cell input count is lower than 1 x 10⁷ cells, adjust biotinylated antibody volume and keep cell density at 1 x 10⁷ cells/mL. Wash volumes can be kept the same as for 1 x 10⁷ cells.

**Adjust buffer volumes to fit the tube you are using.

***If the target expressing cell population is high (e.g., > 2.5 x 10⁶ target cells/mL), increase the amount of beads (maximum double the amount).