Human monocyte and T cell cultures were generated from leukapheresis specimens from consenting donors under IRB-approved protocols. Peripheral blood mononuclear cells (PBMCs) were isolated following density gradient centrifugation (Ficoll-Hypaque, GE Healthcare, Uppsala, Sweden) and cryopreserved. Short-term monocyte cultures were established from thawed PBMCs by plastic adherence for 2 hours at 37° C, in RPMI 1640 medium with 10% heat-inactivated AB serum (Life Technologies, Carlsbad, CA). Non-adherent cells were removed by washing to enrich for adherent monocytes. CD3+ T cells were isolated from PBMCs using the MACS Pan T Cell Isolation Kit II (Miltenyi Biotec, Auburn, CA) and cultured in RPMI 1640 medium with 10% heat-inactivated AB serum and cytokines as described below. The melanoma cell lines 537-mel, 1363-mel, and 1558-mel, established from metastatic melanoma specimens as previously described,11 (link) were cultured in RPMI 1640 medium with 10% heat-inactivated FBS (Life Technologies; or Sigma-Aldrich, St. Louis, MO). All cultures were maintained in a 37°C, 5% CO2 incubator.
Ab serum
Manufactured by Thermo Fisher Scientific
Sourced in Israel
AB Serum is a cell culture supplement derived from the blood of individuals with the universal AB blood type. It is commonly used as a growth factor in cell culture media to support the proliferation and maintenance of a variety of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Cytometric bead array human th1 th2 th17 cytokine kit, by BD (2 mentions)
Ultra leaf anti cd3 ab okt3, by BioLegend (2 mentions)
Ultra leaf anti cd28 ab cd28, by BioLegend (2 mentions)
Aim 5, by Thermo Fisher Scientific (2 mentions)
Lcki inhibitor, by Merck Group (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Roche (2 mentions)
Penicillin, by Roche (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Xvivo 15 media, by Thermo Fisher Scientific (2 mentions)
Aim 5 media, by Thermo Fisher Scientific (2 mentions)
Macs pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Beta counter, by PerkinElmer (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
3h thymidine, by Cytiva (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Gentamycin sulfate, by Sartorius (1 mentions)
12 protocols using ab serum
1
Isolation and Culture of Human Monocytes and T Cells
2
Allogeneic T Cell Proliferation Assay
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Different numbers of DC were cocultured with 100,000 allogeneic T lymphocytes in RPMI containing 5% AB serum, l -glutamine (2 mmol/L), penicillin (50 U/mL), and streptomycin (50 mg/mL; all from Life Technologies). On day 5, 0.5 µCi/0.2 mL [3H]-thymidine (Amersham Pharmacia, Piscataway, NJ, USA) was added. Incorporated radioactivity was quantified after 24 h by means of a beta counter (Perkin Elmer, Gaithersburg, MD, USA).
3
CD8+ T cell activation and IFNγ analysis
4
CD8+ T cell activation and IFNγ analysis
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Isolated CD8+ T cells from PBMCs (see above) were activated in 96-well plates coated with 5mg/ml plate bound Ultra-LEAF™ anti-CD3 Ab (OKT3, Biolegend) and 2mg/ml plate bound Ultra-LEAF™ anti-CD28 Ab (CD28.2, Biolegend) at a density of 1 × 105 cells per well, in a 1:1 mix of Aim 5 (Gibco) and RPMI medium (Gibco), supplemented with 10% AB serum (Life Technologies), 100 U/ml penicillin (Roche), 100 mg/ml streptomycin (Roche). After 2 h, cells were either left untreated or were treated with 5 nM LCKi inhibitor (Merck Milipore) for the indicated time periods. Cells were subsequently washed 3 times with medium to remove already secreted IFNγ, and cells were then cultured for 3h in 40 ml fresh control medium or fresh medium containing 5 nM LCK inhibitor. Subsequently, supernatants were collected and IFNγ concentrations were determined using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit, according to the manufacturer’s protocol.
5
Expansion of Antigen-specific T Cells
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PBMCs were incubated over night with a mixture of relevant peptides at a concentration of 0.5 µM of each peptide in X-vivo 15 (Fisher Scientific) media supplemented with 5% AB Serum (Invitrogen). The cells were subsequently harvested, washed and plated at a concentration of 5 × 106/ml supplemented with 50U/ml IL-2 for expansion. Fresh media and IL-2 were supplemented every second day until the cells were harvested at day 8, and IL-15 (15ng/ml) was added the last four days.
6
PBMC Expansion with Peptide Stimulation
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PBMCs were incubated overnight (37°C, 5% CO2) in X-vivo 15 media (Fisher Scientific) supplemented with 5% AB Serum (Invitrogen) and a mixture of relevant peptides at a concentration of 0.5 μM of each peptide. The cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 × 106/ml supplemented with 50 U/ml IL-2 for expansion. Fresh media and IL-2 were supplemented every second day until the cells were harvested at day 8, and IL-15 (15 ng/ml) was added the last 4 days.
7
Immune response to bacterial pathogens
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CB mononuclear cells were seeded in 96-well U-bottom plate at 105 cells per well in AIM-V media (Life Technologies) supplemented with 2% AB serum (Invitrogen). The number of monocytes was assumed at 10% of total cells. P. aeruginosa, L. monocytogenes, UPEC, S. typhimurium and A. baumanii were added to the culture at a multiplicity of infection 1 per monocyte and incubated overnight. Cell supernatant was collected and analysed for cytokine production with the multiplex assay (Luminex).
8
Activation of T cells and monocytes
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Healthy CB cells were seeded in 96-well U-bottom plate at 2 × 105 cells per well in AIM-V media (Life Technologies) supplemented with 2% AB serum (Invitrogen). rhIL-12p40 (BD) and/or rhIFN-α2 (PBL, Piscataway, NJ) were added either alone or in different combinations of concentrations for 24 h and the activation of T cells and monocytes analysed using FACS (BD). The concentrations of rhIL-12p40 tested were 0.1–1,000 ng ml−1 and 0.0004–4 ng ml−1 for rhIFN-α2.
9
PBMC Expansion via Peptide Stimulation
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PBMCs were incubated overnight (37 o C, 5% CO 2 ) in X-vivo 15 media (Fisher Scientific) supplemented with 5% AB Serum (Invitrogen) and a mixture of relevant peptides at a concentration of 0.5µM of each peptide. The cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 x 10 6 /ml supplemented with 50U/ml IL-2 for expansion. Fresh media and IL-2 were supplemented every second day until the cells were harvested at day 8, and IL-15 (15ng/ml) was added the last four days.
10
PBMC Isolation Using Density Gradient
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PBMCs were isolated by density gradient separation using LymphoPrep (cat #1114547; Axis-Shield, Oslo, Norway) at 1200 x g for 30 minutes, washed twice in phosphate buffered saline followed by centrifugation at 400 x g, and re-suspended in RPMI 1640 (cat #01-106-1a; Biological Industries, Kibbutz Beit Haemek, Israel) containing 30% (v/v) AB serum, 25 mM HEPES, 2 mM L-glutamine (cat #25030–024; Gibco, Life Technologies, Carlsbad, CA), and 50 μg/mL gentamycin sulfate (cat #03-035-1c; Biological Industries). All subsequent centrifugations were carried out at 400 x g. Human serum isolated from healthy male donors of blood group AB (AB serum) was purchased from Lonza (cat # 14-490E; Basel, Switzerland) and used as the serum source in all experiments.
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