Cat. # | Size | Qty. | Price |
---|---|---|---|
48669S | 1 Kit |
|
Product Includes | Quantity (with Count) | ||||
---|---|---|---|---|---|
Lyophilized DNase I | 3 x 1 ea | ||||
Releasable Streptavidin Beads | 1 x 5 ml | ||||
DNase I Reconstitution Buffer | 1 x 2 ml | ||||
Biotinylated G4S Linker (E7O2V) Rabbit mAb | 2 x 1250 µl |
Product Information
NOTE: Kit capacity: ~2 x 109 total input cells
NOTE: Refer to Table 1 for recommended tube sizes and volumes.
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.
NOTE: Thaw immediately before use and keep on ice once thawed. Thawed reconstituted DNase I can be refrozen once.
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.
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).
NOTE: Before transferring released cells to new tube in step 17, pre-coat collection tubes with Cell Elution Buffer for at least 5 min.
Table 1. Recommended Volumes for Different Cell Numbers
Cell Enrichment Step | Step Description | Volumes Per 1 x 107 Total Input Cells | Volumes Per 2 x 108 Total Input Cells |
Recommended tube size | 5 mL | 50 mL | |
Recommended magnet | e.g., Invitrogen DynaMag-5 | e.g., Invitrogen DynaMag-50 | |
1* | Biotinylated mAb | 12.5 µL | 250 µL |
1* | Cell volume | 1 mL | 20 mL |
3** | Wash cells (PBS-BE) | 2 mL | 40 mL |
4 | Resuspend cells (PBS-BE) | 1 mL | 20 mL |
5*** | Add beads | 25 µL | 500 µL |
7 | Optional: Increase volume (PBS-BE) | 1 mL | 20 mL |
10-11** | Wash cells (PBS-B) | 3 x 1 mL | 3 x 20 mL |
13 | Resuspend cells (Cell Elution Buffer) | 400 µL | 8 mL |
14 | Release cells (reconstituted DNase I) | 4 µL | 80 µL |
18 | Collect residual cells (Cell Elution Buffer) | 400 µL | 8 mL |
*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).
Protocol Id: 3164
All Species Expected
Magnetic bead-based immunoaffinity cell enrichment is a gentle purification method that can be leveraged, in part, to facilitate a robust interrogation of the biology of immune cells that are engineered to express CARs. For example, isolation of rare subsets of CAR positive cells from a complex, heterogeneous population of cells presents the opportunity for extensive downstream analyses using single-cell omics assays. In the context of CAR cell engineering, magnetic bead-based immunoaffinity cell enrichment can also be useful in scenarios when delivery of the CAR transgene is inefficient, resulting in a small population of cells expressing the CAR transgene.
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