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.
Human serum
Manufactured by Gemini Bio
Sourced in United States, Canada
Human serum is a cell-free, sterile liquid component of human blood. It is obtained through the process of centrifugation and separation from the cellular components of whole blood. Human serum can be used as a supplement in cell culture media to support the growth and maintenance of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Benzonase, by Merck Group (4 mentions)
Penicillin streptomycin, by Omega Scientific (4 mentions)
Dynabead, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Dmem with 4.5 g l glucose, by Merck Group (3 mentions)
Human t activator cd3 cd28, by Thermo Fisher Scientific (3 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (3 mentions)
X vivo 15 medium, by Lonza (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (3 mentions)
Ficoll paque plus, by GE Healthcare (3 mentions)
Ficoll paque, by GE Healthcare (3 mentions)
X vivo media, by Lonza (3 mentions)
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)
Rpmi 1640 medium, by Omega Scientific (2 mentions)
45 protocols using human serum
1
Characterization of Cytokine-Producing T Cells
2
Asthma Drugs Impact on CCR7 & NKG7
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In order to assess the effects of asthma medications on CCR7 and NKG7 levels, we isolated PBMC from six healthy volunteers consist of three female and three males; All volunteers were Han nationality and the median age was 43years (range 36–48 years). Then, PBMC were cultured in RPMI 1640 medium supplemented with 10% human serum (Gemini Bio-Products) and then treated with 10−5 M dexamethasone for 12 and 24 h. RNA of cell samples was then collected for RT-PCR.
3
Polymer Micelle Size Determination
4
Detecting Antigen-Specific T Cell Activation
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The AIM assay was previously described (31 (link)). This assay detects cells that are activated as a result of antigen-specific stimulation by upregulation of activation-induced surface markers. Here, we employ 4-1BB (CD137) enrichment (CD137 MicroBead Kit, Miltenyi, Germany) followed by staining with a cocktail containing CD4-APC-ef780, CD3-AF700, CD8/CD14/CD19-V500, CD45RA-eF450, CCR7-PerCPCy5.5, 4-1BB-APC, and OX40-PECy7 (see Table S2 in Supplementary Material). Cryopreserved PBMCs were thawed, and 1 × 106 cells/condition were stimulated with LoMo megapool (2 µg/ml) or high molecular weight (PUR) urine extracts (10 µg/ml) in 5% human serum (Gemini Bioproducts) for 24 h. DMSO (0.25%) with medium was used as negative control. For intracellular cytokine staining (ICS), PBMCs were incubated with LoMo megapool or PUR extract for 24 h. After 20 h, BFA [5 μg/ml (BD Bioscience, San Diego, CA, USA)] was added for an additional 4 h. Cells were then washed, stained for extracellular markers for 30 min, washed again, fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin (Sigma), and stained for intracellular IL-4-BV421, IL-5-PE, IL-17-FITC, and IFN-γ-PerCPCy5.5 (Table S2 in Supplementary Material). PMA and ionomycin (1 and 0.1 µg/ml) were used as positive control. Samples were acquired on a BD LSRII Flow Cytometer and analyzed using FlowJo X Software.
5
Maintenance and Differentiation of HEK293T, TZM-bl, and Human Monocyte-Derived Macrophages
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HEK293T and TZM-bl cells were maintained in DMEM with 4.5 g/L glucose (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin in a 37°C and 5.0% CO2 environment. Human monocytes from multiple anonymous normal donors were elutriated as described previously (Gruber et al., 1995 (link)). Elutriated monocytes were obtained from the NIH blood bank under protocol 99-CC-0168: “Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use.” Monocytes (2 × 106 cells/well in a 12-well plate) were cultured in 1 mL of complete DMEM supplemented with 10% pooled human serum (Gemini Bio-Products, West Sacramento, CA, USA) for 5–7 days to allow differentiation into macrophages (MDMs).
6
Phenotypic Analysis of PBMC
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Cryopreserved PBMCs were thawed in RPMI supplemented with 5% human serum (Gemini Bio-Products, West Sacramento, CA, United States), 1% Glutamax (Gibco, Waltham, MA, United States), 1% penicillin/streptomycin (Omega Scientific, Tarzana, CA, United States), and 50 U/ml Benzonase (Millipore Sigma, Burlington, MA, United States). Cells were then washed and counted. 1 million cells were then blocked in 10% FBS for 10 min at 4°C. After blocking, cells were stained with a combination of APCef780 conjugated anti-CD4 (clone RPA-T4, eBiosciences), AF700 conjugated anti-CD3 (clone UCHT1, BD Pharmigen), BV650 conjugated anti-CD8a (clone RPA-T8, Biolegend), PECy7 conjugated anti-CD19 (clone HIB19, TONBO), APC conjugated anti-CD14 (clone 61D3, TONBO), PerCPCy5.5 conjugated anti-CCR7 (clone G043H7, Biolegend), PE conjugated anti-CD56 (eBiosciences), FITC conjugated anti-CD25 (clone M-A251, BD Pharmigen), eF450 conjugated anti-CD45RA (clone HI100, eBiosciences) and eF506 live dead aqua dye (eBiosciences) for 30 min at 4°C. Cells were then washed twice and acquired or sorted on a BD FACSAria flow cytometer (BD Biosciences, San Jose, CA) to measure the frequency of different cell subsets. The gating strategy is shown in Supplementary Figure S1 .
7
Whole Blood Cultures Under Hypoxia and Elevated CO2
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Whole blood was collected from six healthy male volunteers (mean age, 38 ± 9.9 years). None of the volunteers had a history of hematologic disorders, transplantations, cancers, autoimmune diseases, infection, or immunosuppressive drug use. Whole blood was cultured as reported previously.14 (link) Briefly, 2 mL of whole blood were collected from each volunteer. Whole blood samples were cultured with 18 mL Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA; cat. no. R2405) supplemented with 10% human serum (Gemini Bio-Products, Calabasas, CA, USA; cat. no. H91S001) and 1% penicillin-streptomycin solution (Roche, Basel, Switzerland; cat. no. 11074440001) at 37°C in a humidified incubator with 5% CO2/95% air.
Blood samples were randomly divided into four groups: control (exposed to 20% O2 + 5% CO2 for 24 hours), hypoxia + 5% CO2 (exposed to 5% CO2 + 0.2% O2 for 24 hours), hypoxia + 7% CO2 (exposed to 7% CO2 + 0.2% O2 for 24 hours), and hypoxia + 10% CO2 (exposed to 10% CO2 + 0.2% O2 for 24 hours). The partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and pH of the medium were measured at 24 hours (n = 6 per group) using a Blood Gas/Electrolyte Analyzer (Model 5700, Instrumentation Laboratory, San Diego, CA, USA).
Blood samples were randomly divided into four groups: control (exposed to 20% O2 + 5% CO2 for 24 hours), hypoxia + 5% CO2 (exposed to 5% CO2 + 0.2% O2 for 24 hours), hypoxia + 7% CO2 (exposed to 7% CO2 + 0.2% O2 for 24 hours), and hypoxia + 10% CO2 (exposed to 10% CO2 + 0.2% O2 for 24 hours). The partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), and pH of the medium were measured at 24 hours (n = 6 per group) using a Blood Gas/Electrolyte Analyzer (Model 5700, Instrumentation Laboratory, San Diego, CA, USA).
8
Expansion of PBMC by α-synuclein peptide
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Venous blood was collected in anticoagulant (e.g., heparin or EDTA)-containing blood bags or tubes. PBMC were purified from whole blood using Ficoll-PaqueTM density-gradient centrifugation, according to the manufacturer’s instructions (GE Healthcare Bio-Sciences, Pittsburgh, PA). Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO. For in vitro expansion, cryopreserved PBMCs were thawed in RPMI supplemented with 5% human serum (Gemini Bio-Products, West Sacramento, CA), 1% Glutamax (Gibco, Waltham, MA), 1% penicillin/streptomycin (Omega Scientific, Tarzana, CA), and 50 U/ml Benzonase (Millipore Sigma, Burlington, MA). The cells were then washed and viability was evaluated using the trypan blue dye exclusion method. Briefly, at a density of 2 × 106 per mL, the cells were plated in each well of a 24-well plate in the presence of a α-syn peptide pool at a concentration of 5 μg/ml and were incubated in a 37 °C humidified CO2 incubator for 2 weeks. Intermittently, every 3 days, cells were supplied with 10 U/ml recombinant human IL-2.
9
Culturing of Human Cell Lines
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HEK293T and TZM-bl cells were maintained in DMEM with 4.5 g/L glucose (Sigma-Aldrich, St. Louis, MO) that was supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. THP-1 cells were grown in complete RPMI medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The differentiation of THP-1 cells was achieved by growing cells for 48 h in the presence of PMA (100 ng/mL), and this was followed by a resting period of 24 h, as described (42 (link)). Elutriated human monocytes from multiple anonymous normal donors were obtained from the NIH blood bank under protocol 99-CC-0168: “Collection and Distribution of Blood Components from Healthy Donors for In Vitro Research Use”. Monocytes (4 × 106 cells/well in a 6-well plate) were cultured in 2 mL of complete DMEM supplemented with 10% pooled human serum (Gemini Bio-Products, West Sacramento, CA) and 1 mM sodium pyruvate (Thermo Fisher Scientific, Atlanta, GA 30384) for 5 to 7 days to allow for differentiation into macrophages (MDMs).
10
Radiolabeling and Serum Stability of 90Y-3p-C-NEPA and 177Lu-3p-C-NEPA
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Human
serum was purchased from Gemini Bioproducts (#100110). 90Y-3p-C-NEPA and 177Lu-3p-C-NEPA was prepared by the reaction of 3p-C-NEPA
(50 μg/50 μL H2O) with 90Y or 177Lu (5.55 MBq, 150 μCi) in 0.25 M NH4OAc
buffer (pH 5.5), respectively. Completion of radiolabeling was determined
by ITLC, and the resulting complexes 90Y-3p-C-NEPA and 177Lu-3p-C-NEPA were directly
used for serum stability studies without further purification. 90Y-3p-C-NEPA (6.96 MBq, 188 μCi, 130
μL) was added to human serum (660 μL) in a microcentrifuge
tube. 177Lu-3p-C-NEPA (5.40 MBq, 146 μCi,
100 μL) was added to human serum (500 μL) in a microcentrifuge
tube. The stability of the radiolabeled complexes in human serum was
evaluated at 37 °C for 14 days. The serum stability of the radiolabeled
complexes was assessed by measuring the transfer of the radionuclide
from each complex to serum proteins using ITLC (acetonitrile/water
= 3:2 v/v). A solution of the radiolabeled complex in serum (5–16
μL for ITLC) was withdrawn at the designated time point and
evaluated by ITLC. At each of the time points, the percentage of 90Y released from each of the radiolabeled complexes into serum
was assessed by ITLC. The radiolabeled complex 90Y-3p-C-NEPA or 177Lu-3p-C-NEPA was
detected at ∼50 mm from the bottom of the TLC plate, while
the unbound radionuclide 90Y or 177Lu moved
slower (∼30 mm).
serum was purchased from Gemini Bioproducts (#100110). 90Y-3p-C-NEPA and 177Lu-3p-C-NEPA was prepared by the reaction of 3p-C-NEPA
(50 μg/50 μL H2O) with 90Y or 177Lu (5.55 MBq, 150 μCi) in 0.25 M NH4OAc
buffer (pH 5.5), respectively. Completion of radiolabeling was determined
by ITLC, and the resulting complexes 90Y-3p-C-NEPA and 177Lu-3p-C-NEPA were directly
used for serum stability studies without further purification. 90Y-3p-C-NEPA (6.96 MBq, 188 μCi, 130
μL) was added to human serum (660 μL) in a microcentrifuge
tube. 177Lu-3p-C-NEPA (5.40 MBq, 146 μCi,
100 μL) was added to human serum (500 μL) in a microcentrifuge
tube. The stability of the radiolabeled complexes in human serum was
evaluated at 37 °C for 14 days. The serum stability of the radiolabeled
complexes was assessed by measuring the transfer of the radionuclide
from each complex to serum proteins using ITLC (acetonitrile/water
= 3:2 v/v). A solution of the radiolabeled complex in serum (5–16
μL for ITLC) was withdrawn at the designated time point and
evaluated by ITLC. At each of the time points, the percentage of 90Y released from each of the radiolabeled complexes into serum
was assessed by ITLC. The radiolabeled complex 90Y-3p-C-NEPA or 177Lu-3p-C-NEPA was
detected at ∼50 mm from the bottom of the TLC plate, while
the unbound radionuclide 90Y or 177Lu moved
slower (∼30 mm).
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