CAR (G4S Linker) Cell Enrichment Kit (#48669) Datasheet with Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Information

Storage

All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature.
Releasable Streptavidin Beads are supplied as 4 × 10⁸ beads/mL in phosphate buffered saline (PBS), pH 7.4, containing 0.1% Tween and 0.02% sodium azide as a preservative.
Biotinylated G4S Linker (E702V) Rabbit mAb is supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4°C. Do not aliquot the antibody. Do not freeze.

Specificity / Sensitivity

Biotinylated G4S Linker (E7O2V) Rabbit mAb recognizes exogenously expressed levels of scFv-based CARs containing a G4S linker.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing three Gly4Ser repeats and then conjugated to biotin under optimal conditions. Biotinylated antibody endotoxin levels are less than 0.1 EU / μg.

Product Description

The CAR (G4S Linker) Cell Enrichment Kit is designed to positively select cells that are engineered to express a G4S linker-containing single-chain variable fragment (scFv)-based chimeric antigen receptor (CAR) on the cell surface. Using an indirect magnetic bead-based immunoaffinity enrichment protocol, this kit yields highly pure and viable CAR positive cells that are bead-free. Within a heterogeneous population of cells, desired CAR expressing cells are labeled with a biotinylated rabbit monoclonal antibody targeting the G4S linker. Releasable Streptavidin Beads are then incubated with the cell mixture. Bead-bound cells are isolated without columns using a magnet (not included), and then beads are removed from cells using DNase I. Following release from beads, the positively selected fraction of CAR positive cells containing surface-bound biotinylated antibody-streptavidin complexes are available for expansion or use in downstream applications such as flow cytometry and gene expression analyses.

Bead Specifications:
Releasable Streptavidin Beads are paramagnetic polystyrene beads (4.5 μm diameter) coupled to streptavidin via a DNA linker susceptible to cleavage by DNase I. The beads provided in this kit are not recommended for the isolation of phagocytic cells as the beads are likely to be engulfed by these cells.

Background

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

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