Human PBMCs were rested overnight at 37°C in RPMI 1640 medium (catalog# RP-21, Omega Scientific, Inc.) supplemented with 5% human serum (catalog# 100–512, Gemini Bio-Products), 2 mM L-alanyl-L-glutamine (GlutaMAX-I, catalog# 35050061, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (catalog# 400–109, Gemini Bio-Products) and subsequently stimulated with DENV MP (1 μg/ml for individual peptides) or Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation (catalog# 11132D, Thermo Fisher Scientific) for 3 h at 37°C. IFN-γ- and IL-10-producing cells were labeled using IFN-γ Secretion Assay - Detection Kit (PE), human (catalog# 130–054-202, Miltenyi Biotech) and IL-10 Secretion Assay – Detection Kit (APC), human (catalog# 130–090-761, Miltenyi Biotech), respectively, according to the manufacturer’s instructions. Subsequently, PBMCs were stained with anti-human CD3, CD4, CD8, CD14, CD19, CD45RA, and CCR7 (see Table S4 for antibody details). CD4 IL-10−IFN-γ− DN, IL-10+IFN-γ− IL-10 SP and IL-10−IFN-γ+ IFN-γ SP, and IL-10+IFN-γ+ DP cells were sorted using a FACSAria II cell sorter. Data were analyzed using FlowJo.
Rpmi 1640 medium
Manufactured by Omega Scientific
RPMI 1640 medium is a cell culture medium commonly used in biomedical research. It provides a balanced salt solution and essential nutrients to support the growth and maintenance of a variety of cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads human t activator cd3 cd28 for t cell expansion and activation, by Thermo Fisher Scientific (2 mentions)
Ifn γ secretion assay detection kit pe, by Miltenyi Biotec (2 mentions)
Il 10 secretion assay detection kit apc, by Miltenyi Biotec (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Gemini Bio (2 mentions)
Human serum, by Gemini Bio (2 mentions)
Fetal bovine serum (fbs), by Omega Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ficoll paque plus, by Omega Scientific (1 mentions)
Venor gem mycoplasma detection kit, by Merck Group (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Omega Scientific (1 mentions)
Btx square wave electroporator, by Harvard Apparatus (1 mentions)
5 protocols using rpmi 1640 medium
1
Characterization of Cytokine-Producing T Cells
2
PBMC Isolation and Cryopreservation
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Whole blood samples (with heparin) were centrifuged at 1850 rpm for 15 min with breaks off. Subsequently, the upper fraction (plasma) was collected and stored at −80°C. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE). 35 mL of RPMI 1640 medium (RPMI, Omega Scientific) diluted blood was slowly layered on top of 15 mL Ficoll-Paque PLUS. Samples were spinned at 1850 rpm for 25 min with breaks off. Then, PBMC layers were aspirated and two PBMC layers per donor were combined in a new tube together with RPMI. Samples were spinned at 1850 rpm for 10 min with a low break. Cell pellets of the same donors were combined and washed with RPMI and spinned at 1850 rpm for 10 min with breaks off. Finally, PBMCs were counted using trypan blue and a hemocytometer and, after another spin, resuspended in FBS (Gemini) containing 10% DMSO (Sigma-Aldrich) and stored in Mr. Frosty cell freezing container overnight at −80°C. The next day, samples were stored at liquid nitrogen until further use.
3
Prostate Cancer Cell Line Characterization
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In all studies, the human-derived, PSMA-expressing prostate cancer tumor cell line C4-2 was used (courtesy of Dr. George Thalmann, Inselspital Bern, Switzerland). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Omega Scientific) and grown at 37°C and 5% CO2. Cells were monitored for Mycoplasma contamination using the Venor GeM Mycoplasma detection kit (Sigma Aldrich) and authenticated by short tandem repeat sequencing (Laragen). The parental cells were engineered to express firefly luciferase (C4-2-luc) to allow luciferase-mediated bioluminescence imaging to monitor tumor burden, as previously described (41 (link)).
All animal studies were approved by the UCLA Animal Research Committee (approval 2005-090). The mice were housed under pathogen-free conditions with food and water ad libitum and a 12 h–12 h light–dark cycle. Veterinary staff and investigators observed the mice daily to ensure animal welfare.
All animal studies were approved by the UCLA Animal Research Committee (approval 2005-090). The mice were housed under pathogen-free conditions with food and water ad libitum and a 12 h–12 h light–dark cycle. Veterinary staff and investigators observed the mice daily to ensure animal welfare.
4
Characterization of Cytokine-Producing T Cells
5
Jurkat T-cell Transfection Protocol
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The Jurkat T-cell strains Clone E6-1 (Weiss and Stobo, 1984 (link)) and JCaM1.6 (Lck-deficient) (Straus, 1992 (link); Goldsmith and Weiss, 1987 (link)) were gifts from the laboratories of A. Weiss (University of California, San Francisco) and Y. Shimizu (University of Minnesota), respectively. Both cell lines were authenticated by STR profiling and tested negative for mycoplasma (ATCC, Manassas, Virginia). Jurkat cell lines were cultured in RPMI-1640 medium supplemented with 5–10% fetal calf serum (Omega Scientific, Inc, Tarzana, CA) and 2 mM glutamine, penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO) as described previously (Phee et al., 2005 (link)). Jurkat and JCaM1.6 cells were transiently transfected via electroporation, as described previously (Phee et al., 2005 (link)). Briefly, cells were grown overnight in antibiotic-free RPMI-1640 medium supplemented with 10% fetal bovine serum (Omega Scientific) and 2 mM glutamine (RPMI10). Batches of 15 M cells were resuspended in RPMI10 with 10–15 µg plasmid DNA per construct. Cells were rested, electroporated at 285 V for 10 ms in a BTX square-wave electroporator (Harvard Apparatus, Holliston, MA), resuspended in RPMI10, and allowed to recover overnight. One million live cells were then resuspended in phosphate-buffered saline (PBS), rested for 30 min at 37°C, and stimulated.
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