CD3+ T cells were isolated using CD3-immunomagnetic beads (Miltenyi Biotech, Auburn, CA) and CD4+ T cells using negative selection with magnetic beads (Stemcell Technologies, Vancouver, BC, Canada) and were at least 95% pure.
Cd3 immunomagnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD3 immunomagnetic beads are a laboratory product used for the isolation and enrichment of CD3-positive cells from biological samples. The beads are coated with antibodies that specifically bind to the CD3 surface antigen, which is expressed on T lymphocytes. This allows for the selective separation and purification of T cells from complex cell mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions)
Cd3 cd28 stimulation beads, by Thermo Fisher Scientific (2 mentions)
Magnetic beads, by STEMCELL (1 mentions)
Facsaria, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Immunomagnetic beads, by Miltenyi Biotec (1 mentions)
Isotonic percoll, by Cytiva (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Alexa fluor 647 labeled polyclonal goat anti mouse igg h l antibodies, by Affinity Biosciences (1 mentions)
Polybrene, by Merck Group (1 mentions)
X vivo 15 cell medium, by Lonza (1 mentions)
Hgm csf, by Thermo Fisher Scientific (1 mentions)
Af 300 03, by Thermo Fisher Scientific (1 mentions)
Facsvantage se, by BD (1 mentions)
Facscan, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
9 protocols using cd3 immunomagnetic beads
1
T Cell Isolation and Enrichment
2
Isolation of CD3+ T Cells from PBMC
3
CAR-T Cell Expansion and Transduction
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CD3+ T cells were separated from peripheral blood mononuclear cells (PBMCs) with CD3 immunomagnetic beads (Miltenyi) on day 1. The T cells were expanded with CD3/CD28 stimulating beads (Gibco) and IL-2 overnight and then transduced with a lentiviral vector containing a CAR with a CD3-zeta domain to provide a T cell activation signal and a 4-1BB (CD137) domain to provide a co-stimulatory signal (Creative Biolab). Next, the transfection efficiency was detected 5 days after transfection, and the cells were then expanded for another 5 days until the numbers were sufficient for infusion.
4
Isolation of Liver γδT Cells and Hepatocytes
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For isolation of liver γδT cells, whole B6 livers were dissociated using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch-Gladbach, Germany) and CD3+ γδTCR+ T cells were isolated using a presorting step with CD3+ immunomagnetic beads (Miltenyi Biotec) and then sorted by FACS (FACSAria; BD Biosciences, Heidelberg, Germany) while gating on γδTCR+ cells.
For isolation of Gr-1 cells, spleens were dissociated using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch-Gladbach, Germany) and Gr-1+ cells were isolated using immunomagnetic beads (Miltenyi Biotec).
To obtain single cell suspensions of hepatocytes, livers were treated by a four-step perfusion protocol, as we and others have described previously [22 (link)]. The resulting cell suspension was separated by gradient centrifugation with 35% isotonic Percoll (Amersham Biosciences, Piscataway, NJ, USA) at 700× g for 20 min at 20 °C to select for viable single cells.
For isolation of Gr-1 cells, spleens were dissociated using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch-Gladbach, Germany) and Gr-1+ cells were isolated using immunomagnetic beads (Miltenyi Biotec).
To obtain single cell suspensions of hepatocytes, livers were treated by a four-step perfusion protocol, as we and others have described previously [22 (link)]. The resulting cell suspension was separated by gradient centrifugation with 35% isotonic Percoll (Amersham Biosciences, Piscataway, NJ, USA) at 700× g for 20 min at 20 °C to select for viable single cells.
5
Generation of CD19-specific CAR-T cells
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CD3+ T cells from healthy donors were separated from PBMCs using CD3 immunomagnetic beads (#130-097-043, Miltenyi Biotec, Germany), then amplified using CD3/CD28 stimulation beads (#11131D, Thermo Fisher Scientific) and IL-2 (100 IU/mL; Miltenyi Biotec) in X-VIVO 15 medium (Lonza). Cells were activated and expanded for 48 hr followed by transduction 2 hr later with lentivirus. T cells were generally engineered for 9–12 days to express a CD19-specific CAR and stained with Alexa-Fluor 647-labeled polyclonal goat anti-mouse IgG (H+L) antibodies (Affinity) to detect CAR-T cells. All cells were further confirmed by staining with fluorescein isothiocyanate (FITC)-labeled anti-CD3 antibodies (Abcam).
6
Engineered T Cell Activation and Infection
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CD3+ T cells were isolated with CD3 immunomagnetic beads (Miltenyi Biotec) from the patient's peripheral blood mononuclear cells. The amplification was subsequently incubated in RPMI1640 with IL-2 (100 IU/mL), CD3/CD28 activated magnetic beads (Thermo Fisher Scientific), and 10% FBS. After 48 h, the activated T cells were infected with 123CL CAR lentivirus at 37 °C (multiplicity of infection is 5), and the media was increased to 10 mL after 2 h.
7
CAR T Cell Expansion and Transduction
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CD3+ T cells were separated from PBMCs using CD3 immunomagnetic beads (#130-097-043, Miltenyi Biotec, Germany) on day 1. T cells were amplified using CD3/CD28 stimulation beads (#11131D, Thermo Fisher Scientific) and IL-2(100 IU/mL; Miltenyi Biotec) in X-VIVO 15 Cell Medium (Lonza). Cells were then activated and expanded for 48 hours were transduced 2 hr later with the lentivirus (multiplicity of infection is 10) by different temperatures incubation in the presence of polybrene (Sigma) at 8 μg/mL. Then, the cells continue to expand at 37 degrees at an appropriate concentration (0.5-1×106 cells/ml). The transduction efficiency was determined 3 days after transduction. Generally, the T cells were engineered via 9-12 days of manufacturing to express a CD19-specific CAR or CD123-specific CAR.
8
Isolation and Culture of PBMCs
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Human Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of whole blood using Ficoll density gradient centrifugation. Monocytes were isolated by adhesion and differentiated into human monocyte-derived macrophages (hMDMs) for 7 days in RPMI1640 supplemented with hGM-CSF(1000U/ml) (PeproTech AF-300-03). Primary T cells were isolated using CD3-immunomagnetic beads (Miltenyi Biotech, Auburn, CA, USA) and maintained in RPMI1640 supplemented with 10% FBS, 10 U/ml interleukin-2 at 37 °C and 5% CO2. Cells were maintained for further analysis.
9
Intracellular IFN-γ production assay
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The detection method used to evaluate intracellular IFN-γ production was performed as described above. Brie y, in RPMI 1640 medium supplemented with 10% foetal bovine serum, frozen T cells collected previously were suspended and adjusted to a cell concentration of 4 × 10 6 /ml, and 0.5 ml was added to each of the above collected peptide-sensitized autologous DCs, and co-cultured at 37 °C, 5% carbon dioxide (T:DC ratio of 10:1), and the same stimulation was provided three times per week. Recombinant IL-2 (rhIL-2; 20 U/ml) was added every 3 days during the culture, the medium was replaced with fresh medium every 2-3 days, and the cells were expanded according to need. The cells were collected 7 days after the last stimulation, and CD3 immunomagnetic beads (Miltenyi Biotec) were used for positive selection of T cells. The sorted cells were subjected to intracellular cytokine staining analysis and xed in 4% paraformaldehyde until ow-cytometry analysis (FACScan™ or FACSVantage™ SE; BD Biosciences). Flow cytometry data were analysed by CellQuest software (BD Biosciences).
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