Peripheral blood mononuclear cells (PBMCs) (STEMCELL Technologies, Vancouver, Canada) from male healthy donors (25–32 years old) were cultured at 2.25 × 106 cells/ml in complete T cell media: RPMI 1640 (ATCC 30–2001), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate, 1x antibiotic-antimycotic (Gibco), 1x non-essential amino acids (Gibco) and 10 ng/ml recombinant human IL-2 (R&D Systems, Minneapolis, MN). Products were obtained using Institutional Review Board (IRB) approved consent forms and protocols. Cells were stimulated with α-CD3/CD28 Dynabeads (Gibco) for 6 days at a 1:1 ratio of cells:beads unless otherwise noted. After 6 days, viable cells were purified using Lympholyte®-H (Cedar Lane, Burlington, NC). Cells were restimulated for 6 h with α-CD3/CD28 Dynabeads.
Pbmcs
Manufactured by STEMCELL
Sourced in Canada
PBMCs (Peripheral Blood Mononuclear Cells) are a collection of immune cells isolated from peripheral blood. They include lymphocytes (T cells, B cells, and NK cells) and monocytes. PBMCs are widely used in research applications to study immune function and response.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Easysep human t cell isolation kit, by STEMCELL (2 mentions)
Interleukin 2 (il 2), by BioLegend (2 mentions)
Bright glo, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
αcd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 2, by R&D Systems (1 mentions)
Lympholyte h, by Cedarlane (1 mentions)
Fix perm solution, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Hff 1, by American Type Culture Collection (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Peripheral blood mononuclear cells pbmcs, by STEMCELL (1 mentions)
Staurosporin, by Merck Group (1 mentions)
Mda mb 231 breast cancer cells, by European Collection of Authenticated Cell Cultures (1 mentions)
Anti cd3 cd28 coated dynabeads, by Thermo Fisher Scientific (1 mentions)
Lysing buffer, by Lonza (1 mentions)
17 protocols using pbmcs
1
Activation and Purification of Human T Cells
2
Activated PBMC-Mediated Cytotoxicity Assay
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After mixing interleukin-2 (IL-2) (20 IU ml-1) with PBMCs (STEMCELL Technologies, Vancouver, Canada), HNSCC cells (1 × 106) were co-cultured with PBMCs (1 × 107) for 4 days, followed by isolation of the activated PBMCs using Percoll density gradient centrifugation. The HNSCC cells FADU and Cal27 were labeled with CellTrace (Far Red, Invitrogen, Carlsbad, California, USA), and then cultured with activated PBMCs in a medium (STEMCELL Technologies, Vancouver, Canada) containing human CD3/CD28 tetramer antibody complex in polystyrene tube (FALCON, Canada) for 48 h at 37 °C with 5% CO2 in air. Next, the cells were rinsed with PBS, fixed and permeabilized, followed by repair with Fix/Perm solution (BD Biosciences, Franklin Lakes, NJ, USA). The cells were then stained with V450-cleaved caspase 3 antibody (560,627, dilution ratio of 1: 50, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using BD FACS Canto II (BD Immunocytometry Systems, BD Biosciences, Franklin Lakes, NJ, USA). The HNSCC cells were isolated from the HNSCC/PBMC cell mixture by gating for the CellTrace+. Lastly, the activated PBMC-mediated killer HNSCC cells were analyzed by detecting the signal of the V450-cleaved caspase 3 [16 (link)].
3
Culturing Ovarian Cancer and Immune Cells
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Ovarian cancer cell lines were a kind gift from Dr. Ruby Huang (Cancer Science Institute, Singapore). HEK293, HFF1, and IMR90 were purchased from the ATCC. IGROV-1 was cultured in RPMI-1640 with 10% FBS. SKOV3, was cultured in a 1:1 ratio of DMEM (high glucose) and DMEM (low glucose) with 10% FBS added. IMR90, HEK293, and HFF-1 were cultured in DMEM (high glucose) supplemented with 10% FBS and 2 mM L-Glutamine.
Peripheral blood mononuclear cells (PBMCs) were purchased from StemCell Technologies. T cells were isolated from PBMCs using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer’s protocol. In standard culture, T cells were activated with anti-CD3/CD28 Dynabeads (Life Technologies, Grand Island, NY, USA) at a 1:1 bead-to-cell ratio, and cultured in RPMI-1640 supplemented with 10% FBS, and a cytokine cocktail of IL-7 (20 U/mL), IL-15 (10 U/mL), and IL-21 (0.04 U/mL).
Peripheral blood mononuclear cells (PBMCs) were purchased from StemCell Technologies. T cells were isolated from PBMCs using the EasySep™ Human T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada), according to the manufacturer’s protocol. In standard culture, T cells were activated with anti-CD3/CD28 Dynabeads (Life Technologies, Grand Island, NY, USA) at a 1:1 bead-to-cell ratio, and cultured in RPMI-1640 supplemented with 10% FBS, and a cytokine cocktail of IL-7 (20 U/mL), IL-15 (10 U/mL), and IL-21 (0.04 U/mL).
4
Quantifying HIV-1 RNA Induction
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Total CD4+ T cells were isolated from PBMCs (Stem Cell) and cultured (3–6 replicates of 1 million cells/well) in phenol red–free R10 media with or without phorbol 12-myristate 13-acetate (PMA)/ionomycin (Iono) for 7 days in the presence of 300 mM of efavirenz and raltegravir as previously described [21 (link)]. Total induced HIV-1 RNA was quantified in culture supernatants (pooled from replicate wells and tested in duplicate) by qPCR at pre-entry, week 6, and week 12.
5
Isolation and Characterization of Pseudopterosin
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The origin of the extract of pseudopterosin A to D isolated from A. elisabethae (subsequently named PsA-D) was kindly provided by Dr. Russell Kerr (University of Prince Edward Island, Marine Natural Products Lab, Canada) as described in our previous work [16 (link)]. U0126 inhibitor was purchased from Selleckchem (Houston, TX, USA). MDA-MB-231 breast-cancer cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and grown in humidified atmosphere containing 5% CO2 in an RPMI medium. Medium was supplemented with 15% FCS, 100 units·mL−1 penicillin, and 100 µg·mL−1 units streptomycin. PBMCs were purchased from STEMCELL Technologies (Vancouver, Canada) and cultured in the presence of 5% CO2 in RPMI along with 10% FCS, penicillin, and streptomycin. Staurosporin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and medium and antibiotics from Life Technologies (Gibco, Carlsbad, CA, USA).
6
Monitoring MDAMB231 Cell Apoptosis
7
Delivery of Luciferase to PBMCs
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Example 39
Delivery of Luciferase
Human peripheral blood mononuclear cells (PBMCs) (Stemcell Technologies) were transfected with lipid nanoparticles (LNP) encapsulating firefly luciferase (f.luc) circular RNA and examined for luciferase expression. PBMCs from two different donors were incubated in vitro with five different LNP compositions, containing circular RNA encoding for firefly luciferase (200 ng), at 37° C. in RPMI, 2% human serum, IL-2 (10 ng/mL), and 50 uM BME. PBMCs incubated without LNP were used as a negative control. After 24 hours, the cells were lysed and analyzed for firefly luciferase expression based on bioluminescence (Promega BrightGlo).
Representative data are presented in
8
T-cell Activation Modulation by ALCAM
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For our in vitro experiment, T-cell (CD4+CD25−) population was magnetically sorted to exclude the regulatory T-cell population from single-cell suspensions of splenocytes of C57BL/6 mice or human peripheral blood mononuclear cells (PBMCs; STEMCELL Technologies) using CD4+CD25+ T-cell isolation kits (Miltenyi Biotech, CA, USA). The purity of the sorted cells (>95%) was confirmed by flow cytometry analysis prior to the coculture setup. T cells cultured alone served as a negative control. For coculture assays, - CD4+CD25- T cells, stimulated with anti-CD3/CD28-coated Dynabeads (Thermo Fisher) at 1:1 ratio, were cultured on a monolayer of MSCs in the presence or absence of human anti-ALCAM neutralizing antibody (20 µg/mL; R&D Systems, CA, USA) for 24 and 48 h to assess early T-cell activation and Th1 generation, respectively. In a separate set of experiments, cocultures were treated with human anti-CD6 or isotype controls (20 μg/m; R&D Systems) for 24 and 48 h. For murine cocultures, stimulated T cells were cultured alone or on a monolayer of Mock or ALCAM-silenced MSCs.
9
Efficient T Cell Transduction Protocol
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Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood or leukopaks (New York Blood Center). Following red blood cell lysis with ACK (ammonium-chloride-potassium) Lysing Buffer (Lonza), human T cells were isolated from PBMCs (StemCell Technologies) and subsequently activated with 100 IU/mL of IL-2 and Dynabeads Human T-Activator CD3/CD28 at a bead:cell ratio of 1:5 (Thermo Fisher Scientific). Forty-eight hours after initial expansion, T cells were spinoculated with viral supernatant collected from 293Glv9 packaging cells on RetroNectin-coated plates on 2 consecutive days (Takara Clontech). All experiments were normalized for CAR+ viable cells.
10
Quantitative Viral Outgrowth Assay
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Leukapheresis was performed on ART-treated participants. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation with Ficoll (GE Life Sciences). CD4 T cells were enriched from PBMCs (Stemcell Technologies). Cells were serially diluted (2 million, 1 million, 0.5 million, 0.2 million, and 0.1 million cells/well) and plated in 24-well plates, with 12 replicates at each concentration. Phytohemagglutinin (PHA) and irradiated allogenic HIV-negative PBMCs were added to activate the CD4 T cells. CCR5+ MOLT-4 cells [NIH AIDS reagent program (22 (link))] were added 24 hours later for viral replication. Media were changed every 3-4 days and ELISA for p24 protein was performed after 14 days of culture as described in (23 (link)). In general, supernatants were collected from p24+ wells for which fewer than 50% of dilution replicates were positive. To increase the volume of outgrowth virus available for use, viruses were re-grown for 6 days by infecting and proliferating in new HIV-negative CD4+ T cells.
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