Human umbilical CB samples (50–100 mL) were collected in sterile blood packs (SC-200; Terumo Corp., Tokyo, Japan) containing a citrate-dextrose solution as the anticoagulant. Written informed consent was obtained from all mothers before labor and delivery. Protocols for sampling human umbilical CB were approved by our Institutional Review Board, the Clinical Investigation Committee at Tokai University School of Medicine. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. The mononuclear cell layer was collected, washed twice with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS, and resuspended in degassed PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. CD133+ CB cells were separated from 1 × 108 mononuclear cells by a magnetic bead separation method (Miltenyi Biotec, Gladbach, Germany). In brief, CD133+ cells were labeled with a hapten-conjugated monoclonal antibody (mAb) against human CD133 (clone AC133: Miltenyi Biotec), followed by microbeads coupled with an anti-hapten mAb. The bead-positive cells were enriched on positive selection columns twice in a magnetic field. Flow cytometric analysis of purified cells using a phycoerythrin (PE)-conjugated anti-CD133 mAb of a different clone (clone 293C3; Miltenyi Biotec) showed that 95% of the selected cells were positive for CD133.
Magnetic bead separation method
Manufactured by Miltenyi Biotec
Sourced in Germany
The magnetic bead separation method is a laboratory technique used to isolate and purify target cells, proteins, or other biomolecules from a complex mixture. It utilizes magnetic beads coated with specific ligands that can selectively bind to the desired target. The mixture is then placed in a magnetic field, causing the magnetic beads and their bound targets to be separated from the rest of the sample. This method allows for efficient and rapid purification of the target analyte.
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Lab products found in correlation
Anti cd3 cd28 monoclonal antibodies, by Miltenyi Biotec (2 mentions)
Clone 293c3, by Miltenyi Biotec (1 mentions)
Pe conjugated mouse anti human cd34 mab, by BD (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Recombinant human il 7, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 15, by Thermo Fisher Scientific (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Recombinant human interleukin 2 il 2, by Thermo Fisher Scientific (1 mentions)
4 protocols using magnetic bead separation method
1
Isolation and Purification of CD133+ Cells from Human Umbilical Cord Blood
2
Generation of MSLN-Targeted CAR T Cells
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The MSLN CAR construct is a fully human second-generation vector, including the following components in-frame from the 5' end to the 3' end: anti-MSLN single-chain variable fragment (scFv), the hinge and transmembrane domain of the CD8α molecule, 4-1BB co-stimulatory domain (17 (link)), and the CD3 zeta intracellular signaling domain (Figure 1A ). The CD19 CAR was prepared as the control group, and had the same structure, except for anti-CD19 scFv. Lentiviruses were generated from these constructs via transient transfection of HEK293T cells. Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated by gradient centrifugation using mononuclear cell isolation fluid (Oriental Hua Hui, Beijing, China) followed by CD4+ T-cell and CD8+ T-cell positive selection by a magnetic bead separation method (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+ and CD8+ T cells were co-cultured and activated using anti-CD3/CD28 monoclonal antibodies (Miltenyi Biotec) in a 37 ℃, 5% CO2 temperature incubator in vitro for 24 hours, and then transduced with lentivirus (MSLN CAR and CD19 CAR) for 48 hours. After transduction, the CAR T-cells were cultured and expanded in AIM-V medium with interleukin (IL)-2 cytokine (Gibco, Grand Island, NY, USA).
3
Isolation of CD34+ Cells from Human Umbilical Cord Blood
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Human umbilical cord blood samples (70–100 ml each; n = 10) were collected in sterile blood packs containing citrate–dextrose solution as the anticoagulant. After a Ficoll Hypaque (density 1077 g/cm3; GE Healthcare, Norway) density gradient centrifugation, the MNCs were isolated, washed twice, and suspended in sterile PBS with 0.5% BSA and 2 mM EDTA. CD34+ cells were separated from MNCs by a magnetic bead separation method (Miltenyi Biotec, Germany). In brief, the total MNCs were labeled with mouse anti human CD34 mAb coupled with microbeads (MACS). The bead-positive cells (MNC-CD34+) were enriched on positive-selection columns set in a magnetic field. Flow-cytometric analyses of purified cells using a PE-conjugated mouse anti human CD34 mAb (BD Biosciences, USA) showed that 94.1 ± 5.3% of the selected cells were positive for CD34.
4
Generation of Dual-Targeting CAR T Cells
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Healthy donor-derived peripheral blood mononuclear cells were isolated from blood by gradient centrifugation using Lymphoprep™ (Oriental Hua Hui, Beijing, China) followed by CD3+T-cell enrichment by positive selection using a magnetic bead separation method (Miltenyi Biotec, Bergisch Gladbach, Germany). CD3+T cells were cultured and activated in vitro using anti-CD3/CD28 monoclonal antibodies (Miltenyi Biotec) in a 5% CO2 atmosphere at 37 °C for 18–24 h. The activated T cells were then transduced with lentivirus (CD19-CAR, BCMA-CAR and tandem CAR) for 48 h. We also tansduced the BCMA CAR (D1) followed by CD19-CAR (D2) to obtain the T cells expressed two scFv by transduced two lentivirus. After transduction, the CAR T cells were cultured and expanded in a 5% CO2 atmosphere at 37 °C for 14 days in AIM-V medium (Gibco, Grand Island, NY, USA), supplemented with 100 IU/mL recombinant human interleukin-2 (IL-2; Peprotech, Rocky Hill, NJ, USA), 5 ng/ml recombinant human IL-7 (Peprotech), 5 ng/mL recombinant human IL-15 (Peprotech) and 10% autologous plasma [19 ].
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