PBMCs (6 × 106 to 2.4 × 107) from untreated MS patients were stimulated with pools of 5 myelin peptides (final concentration 10 µg/mL each) with recombinant human IL-2 (10 ng/mL) in 96-well plates (5 × 105 cells/well) and incubated (37 °C, 5% CO2) for 1 wk. All T cell culture was done using RPMI 1640 with l -glutamine containing 10% heat-inactivated FBS (Life Technologies), 55 nM 2-mercaptoethanol (Life Technologies), penicillin/streptomycin/glutamine (Life Technologies), 1 mM sodium pyruvate (Life Technologies), MEM nonessential amino acids (Life Technologies), and MEM vitamin solution (Life Technologies). Peptides tested were those indicated in SI Appendix, Table S1 . Wells from individual patient samples were pooled together, CD8 purified, treated with dasatinib, stained with pools of tetramers (containing the same cognate antigens from the primary stimulation), followed by surface staining as detailed above. Tetramer-positive CD8+ T cells were sorted on a FACSAria cell sorter into a 96-well round-bottom plate (50 to 100 cells per well). Sorted cells were stimulated for 2 wk with 2 µg/mL PHA-L (Sigma-Aldrich), 10 ng/mL recombinant human IL-2 (BioLegend), and irradiated (3,000 rad) allogenic feeder PBMCs. Cells were refed with half volume of fresh media and IL-2 every 3 to 4 d.
Pha l
Manufactured by Merck Group
Sourced in United States, United Kingdom, Canada, Germany
PHA-L is a lectin isolated from the red kidney bean (Phaseolus vulgaris). It functions as a cell surface marker to identify and label specific subpopulations of lymphocytes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 2 (il 2), by Thermo Fisher Scientific (6 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Interleukin 2 (il 2), by Merck Group (4 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Human ab serum, by PAN Biotech (3 mentions)
Recombinant human il 21, by Miltenyi Biotec (2 mentions)
Phosphorothioate cpg odn 2006, by InvivoGen (2 mentions)
Protein ladder, by Thermo Fisher Scientific (2 mentions)
Page gel silver staining kit, by Solarbio (2 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (2 mentions)
Hematoxylin, by Solarbio (2 mentions)
Dab substrate kit, by ZSGB-BIO (2 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Lonza (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Vt1000, by Leica camera (2 mentions)
59 protocols using pha l
1
Expansion of Antigen-Specific CD8+ T Cells from MS Patients
2
Recombinant Protein Extraction Protocol
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Recombinant human IL-21 was purchased from Miltenyi Biotech. Histidine-tagged soluble recombinant human CD40 ligand, anti-polyhistidine mAb, and human recombinant a proliferation-inducing ligand (APRIL) were obtained from R&D Systems (Minneapolis, MN, USA). Phosphorothioate CpG-ODN 2006 was purchased from Invivogen (San Diego, CA, USA). PHA-L and PMA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Affini Pure F (ab)2 fragment goat antihuman IgM used for IgM-BCR cross-linking was purchased from Jackson Immunoresearch Laboratories (West Grove, PA, USA). A Mem-PER™ Plus Membrane Protein Extraction Kit from Thermo Scientific (Waltham, MA, USA) was used for membrane extraction.
3
Stimulation of Immune Cells
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Recombinant human IL-21 was purchased from Miltenyi Biotech. Phosphorothioate CpG-ODN 2006 was purchased from Invivogen (San Diego, CA, USA). PHA-L and PMA were purchased from Sigma–Aldrich (St. Louis, MO, USA).
4
Apoptosis Assessment of T Lymphocytes and Leukemia Cells
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The isolated T lymphocytes and human leukemia K562, HL60 and KG1 cells (purchased from Sigma) were maintained in medium containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Merelbeke, Belgium) and cultured at 37 °C in humidified air containing 5% CO2. T lymphocyte cell death was assessed by an annexin-V-FITC/propidium iodide (PI) and annexin-V-APC/7-AAD-based apoptosis detection kit from BD Biosciences according to the manufacturer’s instructions. Cells were seeded in 12-well plates in the presence of 5 μg/mL phytohemagglutinin (PHA-L, Sigma-Aldrich) and 20 U/mL IL-2 (from Sigma). After six days, the cells were harvested, washed twice with PBS-EDTA, stained with annexin-V/PI and analyzed in a FACS machine (NAVIOS-Beckman Coulter, Suarlée, Belgium), and the data generated were analyzed by KALUZA software (Beckman Coulter).
5
Generation of Autologous PHA-Blasts from PBMCs
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Autologous PHA-blasts were generated by stimulating PBMC with 5 µg/mL Phytohemagglutinin-L (PHA-L, Sigma, L2769), followed by culturing in RPMI1640 medium containing 10% FBS and 100 U/mL IL-2 (Gibco, PHC0027) in 24-well plate for about one week12 (link),14 (link),21 (link). At day 3 or 4, cell growth and formation of blasts were assessed by microscopy. After 7 days of culture, PHA-blasts were harvested and frozen down in freezing medium (culture medium + 10% DMSO).
6
T-cell Activation and Cell Culture Protocols
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T-cells were isolated from peripheral
blood samples taken from healthy volunteers aged 18–45 (KCL
ethics approval: HR-18/19-8846) via Ficoll separation.
Blood was slowly added to a 50 mL Falcon tube containing 15 mL of
Ficoll-Paque (GE Healthcare) solution and spun in a centrifuge at
500 g for 25 min. The peripheral blood mononuclear cell layer was
extracted, and the cells were washed twice with PBS and prepared for
activation. Cells were resuspended in RPMI 1640 cell growth medium
at a concentration of 3 × 106 cells/mL and plated
on a 6-well plate (4 mL per well). T-cells were activated with 5 μg/mL
of phytohemagglutinin (PHA-L, Sigma-Aldrich). IL-2 (100 U/mL, PeproTech)
and fresh medium were added every 2–3 days.
Jurkat and
murine myeloma 5T33 (generous gift from Dr. Yolanda Calle-Patino)
cells were cultured in RPMI-1640 medium supplemented with 10% FBS,
200 U/L penicillin, 0.1 g/L streptomycin, and 2 mMl -glutamine.
The cell concentration was maintained in 1 × 105–1
× 106 cells/mL in a humidified chamber containing
5% CO2 at 37 °C.
blood samples taken from healthy volunteers aged 18–45 (KCL
ethics approval: HR-18/19-8846) via Ficoll separation.
Blood was slowly added to a 50 mL Falcon tube containing 15 mL of
Ficoll-Paque (GE Healthcare) solution and spun in a centrifuge at
500 g for 25 min. The peripheral blood mononuclear cell layer was
extracted, and the cells were washed twice with PBS and prepared for
activation. Cells were resuspended in RPMI 1640 cell growth medium
at a concentration of 3 × 106 cells/mL and plated
on a 6-well plate (4 mL per well). T-cells were activated with 5 μg/mL
of phytohemagglutinin (PHA-L, Sigma-Aldrich). IL-2 (100 U/mL, PeproTech)
and fresh medium were added every 2–3 days.
Jurkat and
murine myeloma 5T33 (generous gift from Dr. Yolanda Calle-Patino)
cells were cultured in RPMI-1640 medium supplemented with 10% FBS,
200 U/L penicillin, 0.1 g/L streptomycin, and 2 mM
The cell concentration was maintained in 1 × 105–1
× 106 cells/mL in a humidified chamber containing
5% CO2 at 37 °C.
7
Expansion of CSF-Derived Mononuclear Cells
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CSF-derived mononuclear cells were expanded as previously reported.23 (link),24 (link) Briefly, 2000 cells per well were seeded in 96-well U-botton microtiter plates together with 2 × 105 nonautologous, irradiated PBMC (45 Gy), 1 μg/mL of PHA-L (Sigma, St Louis, MO), and IL-2 supernatant (2000× diluted) (kindly provided by Dr. Sallusto, Institute for Research in Biomedicine, Bellinzona, Switzerland). Medium consisted of IMDM (PAA) containing 100 U/mL penicillin/streptomycin (PAA), 50 μg/mL gentamicin (BioWhittaker, Cambrex, East Rutherford, NJ, USA), 2 mmol/L l -glutamine (GIBCO, Invitrogen, Waltham, MA, USA) and 5% heat-decomplemented human serum (PAA). Additional IL2 was added every 3–4 days. After 2 weeks cells were pooled and analyzed, cryopreserved, or restimulated again with 1 μg/mL PHA, IL-2 and allogeneic irradiated PBMC. We know from our previous experience that this expansion protocol preserves the TCR repertoire.24 (link)
8
Leukocyte Isolation and Staining for Flow Cytometry
9
Immunohistochemistry and Cell Assays
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PHA-L (L2769) was purchased from Sigma (Saint Louis, MO, USA). PAGE Gel Silver Staining Kit (G7210) was purchased from Solarbio (Beijing, China). Cell Counting Kit-8 (CK04) was from Dojindo (Kumamoto, Japan), and CytoTox 96 Non-radioactive Cytotoxicity Assay (G1782) was from Promega (Madison, WI, USA). In Situ Cell Death Detection Kit-TMR red was from Roche Applied Science (12156792910, Mannheim, Germany). Protein ladder was purchased from Thermo (26616), (Vilnius, Lithuania). DAB substrate kit was from ZSBIO (ZLI-9017, Beijing, China), and hematoxylin was from Solarbio (G4070). All the antibodies against CD3E (512415), CD8A (510793), Granzyme B (252579), and Perforin (500093) were purchased from ZEN BIO (Chengdu, China). The A549 (human non-small cell lung cancer cells), Jurkat (human acute T cell leukemia cells), B16-F10 (mouse melanoma cells) and YAC-1 (mouse lymphoma cells) cell lines were from American type culture collection (ATCC).
10
Isolation and Expansion of HLA-E-Restricted T Cells
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HLA-EUL40 T cells were sorted for 5 transplant recipients (#104, #105, #107, #108 and #109) from PBMCs harvested at 12 months post-transplantation as previously described [58 (link)]. Briefly, streptavidin-coated beads (Dynabeads M-280 Streptavidin, Invitrogen, Villebon sur Yvette, France) were saturated with HLA-E/UL4015-23 monomers before incubation with PBMCs (5x106) for 4h. The UL4015-23 peptide corresponding to the own HCMV infecting strain was selected for each patient. HLA-EUL40 T cells were isolated by immunomagnetic sorting and then expanded for 21–30 days as follows: cells were seeded in 96-well plates (3x103/well) and stimulated with phytohemagglutinin (1 μg/mL, PHA-L; Sigma-Aldrich) in the presence of irradiated EBV-transformed B-cell lines and allogeneic PBMC from healthy donors (EFS, Nantes) as feeder. Cells were grown in RMPI-1640 medium supplemented with 8% human serum, 2 mM L-glutamine, 100 U/mL penicillin and 0.1 mg/mL streptomycin and human recombinant IL-2 (150 U/mL). Purity (>95%) of each T cell population was defined after 14 days of culture by tetramer staining.
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