magnetic bead based cell sorting  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Rapid generation of CD19 CAR-T Cells by minicircle DNA enables anti-tumor activity and prevents fatal CAR-B leukemia

doi: 10.1101/2022.11.25.517980

Figure Lengend Snippet: Generation of mcDNA-CD19-CAR-T cells. (a) Schematic of CD19 CAR structure. (b) Schematic of the process to generate CD19-CAR mcDNA in bacterial E.coli strain ZYCY10P3S2T. After L-arabinose induction, pMC-CD19-CAR was separated into mcDNA-CD19-CAR and the bacterial backbone was removed by SceI endonuclease digestion. (c) Human CD8+ T cells were sorted from PBMCs using MACS™. Samples of PBMCs were obtained from three different healthy donors and evaluated by FACS (left), after the sorting process, CD8+ ratio were evaluated by FACS (right). (d) 72 hours after electroporation or lentivirus transfection, the CAR expression of T cells was evaluated by FACS, and non-transfected normal T cells were used as a control (left). (e) Eighteen days after electroporation, CD19-CAR expression was consistently tested by FACS. (f) Proliferation curves of normal T, lentivirus CAR-T, mcDNA-CAR-T cells. The statistical analysis was performed on cells from 3 donors.

Article Snippet: For activation and transduction, CD8+ T cells were sorted from PBMCs using magnetic bead-based cell sorting CD8 MicroBeads (Miltenyi Biotec), the process was performed according to the manufacture’s instruction.

Techniques: Electroporation, Transfection, Expressing, Control