Non-tissue culture treated 24 well plates (BD Biosciences, USA) were coated with 0.5 mL RetroNectin (20 μg/mL) in PBS for 2 hours at room temperature (RT). The RetroNectin solution was aspirated and the wells were blocked with 0.5 mL Hanks' balanced salt solution (HBSS) plus 2% bovine serum albumin (BSA) for 30 minutes at room temperature. The blocking solution was aspirated and the wells were washed with HBSS plus 2.5% HEPES. 0.1 mL of HELA/CAR and control lentiviral supernatants were thawed, diluted rapidly and added to each RetroNectin-coated well. The plates were centrifuged at 3800 rpm for 2 hours at 32 °C (13). The virus-containing supernatant was aspirated from wells. Blood samples were obtained from healthy donors under protocols approved by Jiangsu Province Blood Center. PBMCs were isolated by Ficoll density gradient separation. Subsequently, PBMCs were activated with anti-CD3 (OKT3) and anti-CD28 antibodies-coated plates for 3 days without IL-2. The HELA/CAR lentivirus coated wells were seeded with 1×106 activated T cells at a concentration of 0.5×106 cell/mL in T cell medium (GT-T551, Takara, Japan) plus 100 U/mL of IL-2. After the T cells were added to each well, they were centrifuged at 1000 g for 10 minutes at 32 °C. The plates were incubated at 37 °C overnight. The transduction was repeated the next day.
Gt t551
Manufactured by Takara Bio
Sourced in Japan, China
The GT-T551 is a high-quality laboratory equipment designed for conducting DNA thermal cycling reactions. It features precise temperature control and efficient heat transfer to ensure accurate and reliable results during the PCR (Polymerase Chain Reaction) process.
Automatically generated - may contain errors
Lab products found in correlation
Gt t551, by BD (2 mentions)
Recombinant human il 2, by Novartis (2 mentions)
Retronectin, by Takara Bio (2 mentions)
24 well plate, by BD (1 mentions)
Anti cd3 antibody, by Thermo Fisher Scientific (1 mentions)
Facsaria 1, by BD (1 mentions)
Ficoll density gradient, by GE Healthcare (1 mentions)
Cellquest software, by BD (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Recombinant human il 2, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
8 protocols using gt t551
1
Lentiviral Transduction of Activated T Cells
2
Isolation and Characterization of CIK Cells
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Human peripheral blood samples were obtained from healthy volunteer blood donors of blood (Tianjin Blood Center). PBMCs were isolated by centrifugation on Ficoll density gradients (GE Healthcare Life Sciences, Shanghai, China). To induce CIK cells, PBMCs were incubated in serum-free medium GT-T551 (Takara, Japan) containing 100 ng/mL anti-CD3 antibody (e-Bioscience, San Diego, USA), 100 U/mL rhIL-1α, and 1000 U/mL rhIFN-γ (R&D system, Inc, Minneapolis, MN) on day 1. Subsequently, 200 U/mL rhIL-2 was added to the medium on day 2, and the medium was regularly replaced with fresh IFN-γ- and IL-2-containing medium every 3–5 days. On day 14, cells were harvested and analyzed for the phenotypes of CIK cells by flow cytometric assay. Briefly, the phenotypes of induced CIK cells were detected through flow cytometry using a series of fluorescence-labeled monoclonal antibodies specific for CD3, CD4, CD8, CD25, CD127, CD16/CD56, CD45RA, CD45RO (eBioscience, San Diego, CA). Induced CIK cells (5 × 105) were incubated with these antibodies for 30 min on ice, and then cells were washed twice and analyzed on Fluorescence-Activated Cell Sorting (FACS) Aria I (BD Biosciences, San Diego, CA, USA) by using CellQuest software (BD Biosciences, San Diego, CA, USA).
3
Expansion and Cryopreservation of Human PBMCs
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Human peripheral blood lymphocytes (PBMCs) were isolated from healthy human volunteers, and the informed consents were obtained from all volunteers. Cells were cultured in medium consisting of GT-T551 (TaKaRa) supplemented with 2000 IU/ml recombinant human interferon-gamma (Chemo Wanbang Biopharma, Shanghai, China), 10 mg/ml OK432 (Chugai Pharmaceutical, Chome, Japan), 2% autologous plasma and 1% penicillin-streptomycin on day 1 for 24 h. Then cells were transferred to an anti-CD3 (T&L Biological Technology, Beijing, China) antibody-coated flask in GT-T551 medium supplemented with 700 IU/ml recombinant human interleukin-2 (IL-2) (BD Biosciences) for 14 days. Cells were harvested and stored at −80 °C for further experiments. The protocol of this study was approved by the research ethics committee of Medical School of Nanjing University. The experimental methods were carried out in accordance with the approved guidelines and regulations.
4
Expansion of Activated Human T Cells
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PBMCs were isolated from fresh heparinized venous blood samples using density-gradient centrifugation in Ficoll-Paque PLUS (GE Healthcare) and then were stimulated using plates coated with CD3 (5 μg/mL; OKT3, Janssen Pharmaceutica, Beerse, Belgium) and RetroNectin (25 μg/mL; Takara Bio). The cells were cultured with GT-T551 (Takara Bio) supplemented with 600 IU/mL human recombinant IL-2 (Novartis), 0.2% human serum albumin (CLC Behring, Danville, CA, USA), and 0.6% autologous human plasma. The cells were used for experiments on days 7–11.
5
Serum-free Secretome Analysis
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HCT116-FOXQ1 and DLD1-shFOXQ1 cells were cultured in RPMI-1640/5% FBS medium. At 90% confluence, the culture medium was switched to either serum-free GT-T551 (Takara, Dalian, China) or fresh RPMI-1640/5% FBS medium and was incubated for 48 h before the CM was collected. Serum-free GT-T551 CM was collected for protein array analysis, and RPMI-1640/5% FBS CM was collected for EC migration assay and ELISA.
6
Isolation and Expansion of Human PBMCs
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Human peripheral blood (50 mL) was obtained from healthy male volunteers, and all volunteers have signed the informed consent. Human PBMCs were isolated freshly via density gradient centrifugation using Ficoll (Hao Yang Biological Products Technology, Tianjin, China) as described elsewhere [24 (link),25 (link)]. Cells were then washed with PBS and propagated at 1 × 106 cells/mL in GT-T551 (Takara Bio, Shiga, Japan) containing 2000 IU/mL recombinant human IFN-γ (Chemo Wanbang Biopharma, Shanghai, China), 10 mg/mL OK432 (Shanghai Chugai Pharmaceutical, Chome, Japan), 1% penicillin-streptomycin and 2% heat-inactivated autologous plasma for 24 h. After that, cells were transferred to a cell culture flask with 5 μg/mL anti-CD3 antibody coated (T&L Biological Technology, Beijing, China). Then, GT-T551 medium containing 700 IU/mL recombinant human IL-2 was added (BD Biosciences, CA, USA). After 14 days, cells were detected by flow cytometry, collected and kept at −80 °C for further use. The experiment was approved by the Ethics Committee of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School, China.
7
Preparation of NY-ESO-1-Expressing T Cells
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Human T cells expressing NY-ESO-1 receptor were prepared by retroviral transduction. In brief, PBMCs were stimulated using plates coated with anti-CD3 (5 μg/mL; OKT3, Janssen Pharmaceutica) and RetroNectin (25 μg/mL; Takara Bio) and were cultured with GT-T551 (Takara Bio) supplemented with 600 IU/mL human recombinant IL-2 (Novartis), 0.2% human serum albumin (CLC Behring), and 0.6% autologous human plasma. On days 4 and 5, these cells were transduced with the retroviral vector pMS3 containing NY-ESO-1 using the RetroNectin-bound virus infection method, wherein retroviral solutions were preloaded onto RetroNectin (Takara Bio)-coated plates and centrifuged at 2,000 × g for 2 h at 32°C, followed by expansion culture. The cells were used for experiments on day 11.
8
Ex Vivo Expansion of Cytokine-Induced Killer Cells
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Human peripheral blood samples were obtained from volunteer donors. Fresh peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using cell separation media Lymphoprep (Tianjin Chuanye Biotechnology Co., Ltd. Tianjin, China). PBMCs were cultured in GT-T551 (Takara, Japan) containing with 10% FBS (Sigma, USA) in 1000 U/ml human interferon (IFN)-γ1b (Miltenyi Biotec, Auburn, USA) overnight. After 24 h in culture at 37 °C and 5% CO2, 50 ng/ml LEAFTM purified anti-CD3 antibody (Biolegend, San Diego, USA) and 300 U/ml recombinant human interleukin (IL)-2 (Shandong Quangang Pharmaceutical Co. Ltd, Shandong, China) were added. Fresh medium with IL-2 was added as needed. Finally, the expanded bulk CIK cells were analyzed by flow cytometry.
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