CD4+ T cells of PBMC were isolated using the Human CD4 Positive Selection Kit II (STEMCELL, 17852). Purified CD4+ T cells were conjugated with 2.5 mM CFSE (BD Biosciences) for 10 min at 37 °C while mixing continuously. Chilled complete growth media was added to quench the labeling reaction. Subsequently, the cells labeled with CFSE were stimulated with CD3/28 Dynabeads (GIBCO, Grand Island, NY, USA) or CD3/28 Dynabeads plus SMI-4a (20 μM). After incubation for 72 h, the proliferation rate of CD4+ T cells was measured using flow cytometry. Similarly, B cells sorted from PBMCs were conjugated with 2.5 mM CFSE. The cells labeled with CFSE were stimulated with CD40L and IL-4, or CD40L and IL-4 plus SMI-4a (20 μM). After incubation for 4 days, the proliferation rate of B cells was assessed using flow cytometry.
Cd3 28 dynabeads
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD3/28 Dynabeads are superparamagnetic beads coated with antibodies specific to the CD3 and CD28 surface receptors on T cells. These beads are designed to activate and expand T cells in vitro for various research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by BD (2 mentions)
Alexa fluor 700, by Thermo Fisher Scientific (1 mentions)
Optmizer media, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Leukopak, by AllCells (1 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (1 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd3 dynabeads, by Miltenyi Biotec (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay kit, by Promega (1 mentions)
Facscalibur flow cytometer, by Keygen Biotech (1 mentions)
Annexin 5 and pi, by Keygen Biotech (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Human na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Polybrene reagent, by Merck Group (1 mentions)
10 protocols using cd3 28 dynabeads
1
CFSE-Based T and B Cell Proliferation Assay
2
CFSE-Labeled CD4+ T Cell Activation
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Human CD4+ T cells were obtained using flow sorter and stained with 2.5 mM carboxyfluorescein diacetate succinimidyl ester (CFSE, BD Biosciences, Franklin Lakes, NJ, USA). Then, these cells were treated with CD3/28 Dynabeads (GIBCO, Grand Island, NY, USA) or CD3/28 Dynabeads+ KC7F2 (10uM) for 3 days and subjected to flow cytometry.
3
CFSE-based T Cell Proliferation Assay with MSCs
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CD3+ T lymphocytes were isolated from umbilical cord blood samples (Canadian Blood Services, Montreal, QC, Canada) using a positive selection kit (StemCell Technologies, Vancouver, BC, Canada) and stained with 1 µM CFSE (Molecular Probes, Thermo Fisher, Burlington, ON, Canada) for 5 min. Cells were washed twice with PBS (Invitrogen, Thermo Fisher, Burlington, ON, Canada), resuspended in OpTmizer media (Invitrogen), and 4 × 104 cells per well were seeded in triplicate in a 96 wells plate (Life Science, Corning, Tewksbury, MA, USA) and stimulated with CD3/28 Dynabeads (Invitrogen). MSCs were grown as described above in proinflammatory conditions induced with TNFα and IFNγ, 48 h prior to harvesting. MSCs were resuspended in OpTimizer media and 4 × 103 cells were added to the stimulated T cells. Co-cultures were placed at 37 °C in a humidified incubator, at 5% CO2 and 5% O2, for five days. To analyze T lymphocyte proliferation, cells were stained with anti-CD45 antibodies (clone 2D1) conjugated to AlexaFluor700 (eBioscience, Thermo Fisher, Burlington, ON, Canada) and 10 ng µL−1 propidium iodide (Sigma) and analyzed using a BDFortessa flow cytometer. Stimulated CFSE fluorescence of CD3+CD45+ cells, in the absence or presence of MSCs, were compared to determine T cell proliferation.
4
Lentiviral Transduction of T Cells
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A*02:01À peripheral blood mononuclear cells (PBMCs) from four donors were isolated from fresh Leukopak (AllCells) using Ficoll density gradient media. CD4+ T cells or Tregs were isolated using a CD4+ T-cellÀ isolation kit or CD4+CD25+ regulatory T-cell isolation kit (Miltenyi) according to the manufacturer's instructions. Post isolation, T cells were cultured at a density of 1 £ 10 6 cells/mL in X-Vivo 15 media supplemented with 5% human A/B serum and 1% penicillin/ streptomycin in the presence of CD3/28 Dynabeads (Invitrogen) and 300 units/mL of IL-2 (Miltenyi). After 2 days, cells were transduced with lentivirus (outsourced) at a multiplicity of infection of 5. Cells were cultured in IL-2 for an additional 5 days prior to enrichment.
5
Quantitative Profiling of CAR-T Cells
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Post-treatment T cells were either stimulated by incubating for 5 h in the presence of CD3/28 Dynabeads (Gibco) to induce non-specific stimulation or left unstimulated through the time-period. The T cells were then washed with PBS and resuspended in 10 μL RLT buffer + β-mercaptoethanol. RNA from the lysed cells was amplified using CT1000 Touch Thermal Cycler (Bio-Rad) and hybridized with the default probes in the nCounter CAR-T Characterization Panel. A fully automated nCounter Prep station liquid-handling robot was used to process the hybridized sample into the nCounter cartridge. The nCounter cartridge was then placed into the nCounter digital analyzer for direct digital counting according to the manufacturer’s protocol. The counts were then exported and analyzed.
6
Isolation and Transduction of T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors by Ficoll-Paque density centrifugation. Then, T cells were isolated from PBMCs using magnetic cell separation (MACS) and CD3 Dynabeads (Miltenyi Biotec, Germany) and cultured in X VIVO-15 medium (LONZA, Switzerland) supplemented with 200 IU/ml IL-2 and CD3/28 Dynabeads (Gibco, USA).
T cells were transduced by incubation with a CAR lentivirus (CAR-T) or vector lentivirus (Control T). After immediate centrifugation for 90 min at 37 °C, the transduced cells were cultured at 37 °C and 5% CO2 for 48 h. LMP1-CAR-EGFP fusion vector was used for the detection of transfection efficiency and in vitro cytotoxicity assay. LMP1-CAR vector without EGFP was used for the detection of T cell activation markers, cytokine production and in vivo experiments. The transfection efficiency of CD38-CAR or two Tan CAR-T cells was detected by fluorescence-activated cell sorter (FACS) using fluorescein isothiocyanate (FITC)-labelled recombinant human CD38 protein (ACROBiosystems, China).
T cells were transduced by incubation with a CAR lentivirus (CAR-T) or vector lentivirus (Control T). After immediate centrifugation for 90 min at 37 °C, the transduced cells were cultured at 37 °C and 5% CO2 for 48 h. LMP1-CAR-EGFP fusion vector was used for the detection of transfection efficiency and in vitro cytotoxicity assay. LMP1-CAR vector without EGFP was used for the detection of T cell activation markers, cytokine production and in vivo experiments. The transfection efficiency of CD38-CAR or two Tan CAR-T cells was detected by fluorescence-activated cell sorter (FACS) using fluorescein isothiocyanate (FITC)-labelled recombinant human CD38 protein (ACROBiosystems, China).
7
Cytotoxicity Assessment of CD38-CAR-T Cells
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CD38‐CAR‐T or control T cells (effector) were cocultured with MM cell lines (target cells) at E:T (effector to target) ratio of 2:1, 1:1, 1:2, and 1:4 for 6 h. Then the cytotoxicity of CAR‐T cells was determined by measuring lactate dehydrogenase (LDH) in the supernatant. LDH was detected by CytoTox 96® Non‐Radioactive Cytotoxicity Assay kit (Promega).
To evaluate the cytotoxicity of CAR‐T cells at lower E:T ratios, T cells were activated by CD3/28 dynabeads (Gibco) for 3 days and then transduced with CD38‐CAR lentivirus. MM cell lines were labeled by CFSE and then cocultured with CD38‐CAR‐T or control T cells at a serial E:T ratios of 1:10, 1:20, 1:50, and 1:100 for 24 h. Then cells were stained with Annexin V and PI (Keygen Biotech) and analyzed by a FACS Calibur flow cytometer. Cytotoxicity of CD38‐CAR‐T cells against autologous PBMCs and primary tumor cells isolated from bone marrow in MM patients was also evaluated. PBMCs or primary tumor cells were co‐incubated with CAR‐T and control T cells at a E:T ratio of 1:1 or 1:10 for 24 h and analyzed by a FACS Calibur flow cytometer.
To evaluate the cytotoxicity of CAR‐T cells at lower E:T ratios, T cells were activated by CD3/28 dynabeads (Gibco) for 3 days and then transduced with CD38‐CAR lentivirus. MM cell lines were labeled by CFSE and then cocultured with CD38‐CAR‐T or control T cells at a serial E:T ratios of 1:10, 1:20, 1:50, and 1:100 for 24 h. Then cells were stained with Annexin V and PI (Keygen Biotech) and analyzed by a FACS Calibur flow cytometer. Cytotoxicity of CD38‐CAR‐T cells against autologous PBMCs and primary tumor cells isolated from bone marrow in MM patients was also evaluated. PBMCs or primary tumor cells were co‐incubated with CAR‐T and control T cells at a E:T ratio of 1:1 or 1:10 for 24 h and analyzed by a FACS Calibur flow cytometer.
8
Naïve CD4+ T Cell Activation Assay
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For purified human naïve CD4+ T cells, a human naïve CD4+ T cell isolation kit was used according to the manufacturer’s instructions (Miltenyi Biotec). Human naïve CD4+ T cells (1 × 105 cells) and SH-10-TC cells (3 × 104 cells) were cultured at 37°C in RPMI 1640 (Lonza) containing 10% fetal bovine serum (Heat inactivated; GeminiBio), penicillin/streptomycin, and 50 μM 2-mercaptoethanol (Sigma Aldrich) in the presence of CD3/28 Dynabeads (Thermo-Fisher Scientific) for 48 h in the presence or absence of 5 μM GO-Y022 or 5 mM 2DG.
9
T-Cell Activation and IFNγ Release
10
Overexpression of miR-150 and Foxo1 shRNA in T cells
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For overexpression of miR-150 and Foxo1 shRNA, MSCV-IRES-Thy1.1 and pMKO.1-Thy1.1 retroviral vectors were provided by Rafi Ahmed (Emory Vaccine Center). pMKO.1 Thy1.1 was cloned with Foxo1 shRNA as described previously (Harada et al., 2010) . MSCV-IRES-Thy1.1 was cloned with miR-150 containing the flanking UTR (200 bp) or the Foxo1-coding sequence from the vector purchased from Addgene (Cambridge, MA, USA). For transduction of retroviral genes, isolated CD8 + T cells from WT, miR-150-KO, or WT P14 mice were stimulated with CD3/28 Dynabeads and infected with LCMV Arm (GIBCO; Thermo Fisher Scientific) for 6 hr or 12 hr, respectively. Activated CD8 + T cells were treated with recombinant retrovirus and mixed thoroughly by pipetting with polybrene reagent (8 mg/mL; Sigma-Aldrich). Cells were centrifuged at 1,800 3 g for 90 min at 37 C to transduce CD8 + T cells. After centrifugation, the supernatant was removed, and cells were washed with RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS). Cells were suspended and used for various applications.
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