A blood sample from the fasting ulnar vein (10 ml) was drawn from each individual and collected in vacutainer tubes containing preservative-free heparin. Next, PBMCs were isolated using standard density centrifugation (2,500 rpm for 30 min at 25°C without a break) with Ficoll-Paque PLUS medium (d=1.077; Amersham Pharmacia Biotech, Uppsala, Sweden). Red blood cell lysis buffer (BioLegend, San Diego, CA, USA) was used to remove residual red blood cells, and the platelets were then removed by centrifugation (1,000 rpm for 15 min at room temperature). Finally, the purification of the T lymphocytes was performed with a T cell enrichment column (R&D Systems, Minneapolis, MN, USA), and the lymphocytes were kept at −70°C until further analysis. According to a flow cytometry analysis (BD Biosciences, San Jose, CA, USA), the purity of the T lymphocytes was >92% after staining with monoclonal antibodies for human CD3 antigens PE (BD Biosciences). The entire procedure was completed within 4 hours of drawing the blood samples.
T cell enrichment column
Manufactured by R&D Systems
Sourced in United States
The T cell enrichment column is a laboratory equipment designed to isolate and concentrate T cells from a mixed cell population. The column utilizes a proprietary matrix to selectively bind and retain T cells, while allowing other cell types to pass through. This allows for the efficient separation and purification of T cells for various applications in research and cell-based therapies.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Macs cell separation, by Miltenyi Biotec (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Easysep magnetic separation system, by STEMCELL (3 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Seradigm 1500 500 fbs, by Avantor (2 mentions)
Anti cd3, by BioLegend (2 mentions)
Mitomycin c, by Merck Group (2 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Retronectin coated, by Takara Bio (1 mentions)
Untouched cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Platinum e plat e retroviral packaging cells, by Cell Biolabs (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Platinum e, by Cell Biolabs (1 mentions)
Pcl neo mova, by Addgene (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
23 protocols using t cell enrichment column
1
Isolation and Purification of T Lymphocytes
2
Murine Anti-CD19 CAR T Cell Generation
3
Isolation and Cultivation of Diverse Immune Cells
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Jurkat T cells (TIB-152), HEK293T (CRL-1573), Raji B cells (CCL-86; all from ATCC), and Platinum-E (Plat-E) retroviral packaging cells (Cell Biolabs, San Diego, CA, USA) were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). Naive CD3+ T cells were purified from the mouse spleen and lymph nodes by negative selection using a T cell enrichment column (R&D Systems). To generate mouse T cell blasts, CD3+ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured for a further 5 days with 100 U/mL rIL-2. Mouse splenocytes were dispersed and purified into CD4+, CD8+, and CD19+ populations using the EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada) or MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. To establish B cell blasts, CD19+ cells from C57BL/6 wild-type mice were activated with LPS (10 μg/mL) for three days in complete RPMI. Bone marrow was flushed from femur and tibia bones, and bone-marrow-derived DCs (BMDCs) were grown with the addition of 20 ng/mL granulocyte macrophage-colony stimulating factor for five days.
4
Isolation and Activation of Immune Cell Populations
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Jurkat T (TIB-152), HEK293T (CRL-1573), and B16F10 (CRL-6475) cell lines were purchased from ATCC. Adult leukemia cell lines, MT2, and MT4 were purchased from CellBank Australia (Westmead, NSW, Australia). The retroviral ecotrophic packaging cell line Platinum-E was purchased from Cell Biolabs (San Diego, CA, USA). Cells were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). A stable B16F10 cell line expressing membrane-bound OVA (B16F10-OVA) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cambridge, MA) using Lipofectamine 2000 reagent (Invitrogen) and selected with G418 (InvivoGen; San Diego, CA, USA). Naïve CD3+ T cells were purified from mouse spleen and lymph nodes by negative selection using a T-cell enrichment column (R&D Systems). Naïve CD4+ and CD8+ T cells and CD11C+ dendritic cells (DCs) were purified from mouse spleen and lymph nodes by negative selection using an EasySep magnetic separation system (Stemcell Technologies; Vancouver, Canada). To generate mouse T-cell blasts, isolated T cells were incubated in 2 µg/ml anti-CD3/28-coated culture plates with 100 U/ml rIL-2 for 48 h and cultured for an additional 3 days with 100 U/ml rIL-2. The purity of each population was confirmed to be more than 95% by flow cytometry.
5
Isolation and Characterization of Mouse Immune Cells
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Mouse thymocytes were purified from the mouse thymus. Mouse splenocytes and lymphocytes were dispersed and purified to CD4+, CD8+, CD19+ and CD11c+ populations by MACS cell separation (Miltenyi Biotec). Naïve CD3+ T cells were purified from the mouse spleen and lymph nodes by negative selection using a T cell enrichment column (R&D Systems). To generate mouse T cell blasts, naïve CD3+ T cells were incubated in 2 μg/ml anti-CD3/28-coated culture plates with 100 U/ml rIL-2 for 48 h and cultured for a further 5 days with 100 U/ml rIL-2. Mouse splenocytes were dispersed and purified into CD4+, CD8+ and CD19+ populations using the EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada) or MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. HEK293T cells (CRL-1573, ATCC), B16F10 cells (CRL-6475, ATCC) and EO771 cells (CRL-3461, ATCC) were maintained in DMEM supplemented with 10% FBS, penicillin and streptomycin.
6
Isolation and Characterization of Immune Cells
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Jurkat T-cells (TIB-152), A7r5 (CRL-1444) cells, and RAW 264.7 cells (TIB71™) were maintained in RPMI-1640 or DMEM (Invitrogen) supplemented with 10% (v/v) FBS (Invitrogen). Mouse CD3+ T-cells were purified from the mouse spleen and lymph nodes on a T-cell enrichment column (R&D Systems). Mouse splenocytes were dispersed and purified into CD4+, CD8+, and CD19+ populations by MACS cell separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of each population was confirmed as >95% by flow cytometry. Mouse brain lysate was obtained from C57BL/6 wild-type mice. Peritoneal macrophage isolations were performed 60 h after an intraperitoneal injection of 2.5 mL 3% thioglycollate solution (Sigma) by peritoneal lavage with 5 mL of PBS. Withdrawn cells were cultured for 1 h at 37 °C and then rinsed to remove suspended cells. Of these adherent cells, >99% were F4/80+ macrophages. To prepare conditioned media for differentiation of BMDMs, L929 cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator. Cell culture media were collected and filtered using 0.22-μm filters and kept at −20 °C. Mouse bone marrow cells were isolated from femur and tibia and cultured in RPMI-1640 supplemented with 30% of L929 cell-conditioned medium. After 6 days, BMDMs were confirmed to be >90% by flow cytometry with CD11b+ and F4/80+.
7
Isolation and Activation of Mouse and Human T Cells
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Spleens and lymph nodes (inguinal and axillary) excised from euthanized mice were homogenized into a single cell suspension followed by CD3+ T cell isolation using a Mouse Pan T cell isolation kit (Miltenyi Biotec), with isolated cells counted using a Millipore Scepter. For human T cells, whole blood was obtained from healthy volunteers as approved by the University of Hawaii's institutional review board, and a T cell enrichment column (R&D Systems) was used for T cell isolation. Mouse and human T cells were activated through the T cell receptor (TCR) in 96-well plates precoated with BioLegend anti-CD3 (clones 145-2C11 and OKT3; 10 μg/mL) plus anti-CD28 (clones 37.51 and CD28.2; 1 μg/mL). Cells were incubated for different periods in RPMI-1640 media containing 10% Seradigm 1500-500 FBS (VWR). In some cases, the expression of SELENOI shRNA was induced in vivo by i. p. injecting DOX at 5 μg/g per day for 2 d prior to spleen/lymph node harvest. Ex vivo T cells from these mice were cultured in complete media containing 2 μg/mL of DOX for continued KD of SELENOI. Images of proliferating T cells were captured on a Zeiss Axiovert 200M attached to a Zeiss LSM 5 Pascal imaging system.
8
Cell Culture and T Cell Activation
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Jurkat T (TIB-152), A7r5 (CRL-1444), HEK293T (CRL-1573), COS-7 (CRL1651), and HeLa (CCL2) cells were maintained in RPMI-1640 or DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) FBS (Invitrogen). Mouse CD3+ T cells were purified from the mouse spleen and lymph nodes on a T-cell enrichment column (R&D Systems). To generate mouse T cell blasts, CD3+ T cells were incubated in 2 µg/mL anti-CD3-coated coverslips with 2 µg/mL anti-CD28 and 10 µg/mL rIL-2 for 48 h.
9
Isolation and Characterization of Primary Immune Cells
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Jurkat T cells (TIB-152; ATCC), A7r5 (CRL-1444; ATCC), HEK293T (CRL-1573; ATCC), CHO-K1 (10061; Korean Cell Line Bank) cells, EL4 (TIB-39; ATCC), and Raji B cells were maintained in RPMI 1640 medium or DMEM medium (Gibco/Invitrogen) supplemented with 10% (vol/vol) FBS (Gibco/Invitrogen). After written informed consent, human primary PBLs were isolated from healthy donors using dextran sedimentation and centrifugation through a discontinuous Ficoll gradient (GE Healthcare). Mouse CD3+ T cells were purified from the mouse spleen and lymph nodes on a T cell enrichment column (R&D Systems). Mouse splenocytes were dispersed and purified to CD4+, CD8+, and CD19+ populations by MACS cell separation (Miltenyi Biotec). The purity of each population was confirmed to be >95% by flow cytometry.
10
MUC1 CAR T Cell Generation
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Mouse T cells from naïve C57BL/6 mice or MUC1.Tg mice were enriched from pooled spleens using a T-cell enrichment column (R&D systems, Minneapolis, MN). CD8+ T cells were further isolated using beads-based negative selection (Miltenyi Biotec). Enriched T cells or isolated CD8+ T cells (1×106 cells/mL) were activated with anti-mouse CD3/CD28 beads (Thermo Fisher Scientific). On day 2, non–tissue culture–treated 6-well plates were coated with Retronectin (Takara) at 4°C overnight. On day 3, Retronectin-coated plates were blocked with 2% bovine serum albumin. Then retroviral supernatants or medium control were added into blocked plates. The T-cell transduction process was referred to by Ma et al.29 (link) The transduction efficiency was determined by its binding to biotinylated human MUC1 peptide. Cells were passaged and expanded as needed and used 3–5 days after initial viral transduction (with ~35%–40% CAR positivity). Activated but nonretroviral transduced cells were included as mock T-cell controls. For in vitro functional assays, MUC1 CAR T cells were generated from enriched T cells. For in vivo mouse tumor treatment, MUC1 CAR T cells were generated from isolated CD8+ T cells in the main text.
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