Fresh tumor samples from surgical resection were cut into ≈1 mm3 pieces, and then single‐cell suspensions from human tumor tissues were generated. Dead cells were eliminated using a Dead Cell Removal Kit (Miltenyi Biotec). Tumor cells were purified with EpCAM+ microbeads (Miltenyi Biotec). Tumor‐specific CTLs within tumors were enriched and purified using anti‐CEA Pentamer‐PE (ProImmune) from HLA‐A2+ patients with CRC expressing CEA. Peripheral blood mononuclear cells (PBMCs) were isolated according to the previously reported methods.[44 ] CD8+ T‐cells were purified using a human CD8+ T‐cell isolation kit (Miltenyi Biotec), and then a complete X‐VIVO‐15 medium (Lonza Walkersville) was used for the ex vivo culture. The cell populations were confirmed to be ≥95% pure by flow cytometric analysis.
Complete xvivo15 medium
Manufactured by Lonza
Complete XVivo15 medium is a cell culture medium designed for the growth and maintenance of a variety of cell types, including hematopoietic, mesenchymal, and immune cells. It is a serum-free, protein-free, and chemically defined formula that provides the necessary nutrients and growth factors for cell proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 cd28 magnetic dynabeads cts, by Thermo Fisher Scientific (2 mentions)
P3 buffer, by Lonza (2 mentions)
Easysep magnet, by STEMCELL (2 mentions)
Easysep human t cell isolation kit, by STEMCELL (2 mentions)
Il 15, by R&D Systems (2 mentions)
Interleukin 7 (il 7), by R&D Systems (2 mentions)
Human cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Epcam microbeads, by Miltenyi Biotec (1 mentions)
Anti cea pentamer pe, by ProImmune (1 mentions)
Dead cell removal kit, by Miltenyi Biotec (1 mentions)
3 protocols using complete xvivo15 medium
1
Tumor-Specific CTLs Isolation Protocol
2
T-cell Activation and Genome Editing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leukapheresis products from anonymous healthy human donors were purchased from STEMCELL Technologies, Inc. and isolated using an EasySep human T-cell isolation kit (Cat #17951). Isolated CD3+ T cells were activated at 1 × 106 cells/ml in a 1:1 ratio with CD3/CD28 magnetic dynabeads (CTS, Thermo Fisher Scientific), 100 U/ml IL-7 and 10 U/ml IL-15 (R&D Systems) for 48 h in complete XVivo15 medium (Lonza) (5% FBS, 50 μM 2-mercaptoethanol, 10 mM N-acetyl l -cysteine). Following the 48-h activation period, CD3+ T cells were debeaded with an EasySep magnet (STEMCELL Technologies, Inc.) prior to resuspension and electroporation at 0.5–1 × 106 cells/ml in P3 buffer (Lonza). Following electroporations with RNPs and HDR templates, fresh medium and cytokines were added every 2–3 days.
3
Isolation and Activation of CD3+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leukapheresis products from anonymous healthy human donors were purchased from STEMCELL Technologies, Inc. and isolated using an EasySep human T cell isolation kit (Cat #17951). Isolated CD3+ T cells were activated at 1x10 6 cells mL -1 in a 1:1 ratio with CD3/CD28 magnetic dynabeads (CTS, Thermo Fisher Scientific), 100 U mL -1 of IL-7, and 10 U mL -1 IL-15 (R&D Systems) for 48 hours in complete XVivo15 medium (Lonza) (5% fetal bovine serum, 50 µM 2-mercaptoethanol, 10 mM N-alcetyl L-cysteine).
Following the 48-hour activation period, CD3+ T cells were debeaded with an EasySep magnet (STEMCELL) prior to resuspension and electroporation at 0.5-1x10 6 cell mL -1 in P3 buffer (Lonza). Following electroporations with RNPs and HDRTs, fresh medium and cytokines were added every 2-3 days.
Following the 48-hour activation period, CD3+ T cells were debeaded with an EasySep magnet (STEMCELL) prior to resuspension and electroporation at 0.5-1x10 6 cell mL -1 in P3 buffer (Lonza). Following electroporations with RNPs and HDRTs, fresh medium and cytokines were added every 2-3 days.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!