Venous blood was collected in heparin-containing blood bags or tubes. Peripheral blood mononuclear cells (PBMC) were purified from whole blood by density-gradient centrifugation, according to the manufacturer’s instructions. Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO. Culturing of PBMCs for in vitro expansion was performed by incubating in RPMI (Omega Scientific) supplemented with 5% human AB serum (Gemini Bioscience), 1% GlutaMAX (Gibco), and 1% penicillin/streptomycin (Omega Scientific) at 2 × 106 per mL in the presence of individual peptide pools at 5 μg/ml. Every 3 days, 10U/ml IL-2 in media were added to the cultures.
Roswell park memorial institute (rpmi)
Manufactured by Omega Scientific
RPMI is a common cell culture medium used for the growth and maintenance of a variety of mammalian cell types, including human and animal cells. It provides the necessary nutrients, vitamins, and other components required for cell proliferation and survival in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Omega Scientific (5 mentions)
Antibacterial antimycotic, by Thermo Fisher Scientific (2 mentions)
Ack lyses buffer, by Quality Biological (2 mentions)
Roswell park memorial institute (rpmi), by Quality Biological (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Omega Scientific (2 mentions)
M csf, by R&D Systems (2 mentions)
7 protocols using roswell park memorial institute (rpmi)
1
Expansion of PBMCs via Peptide Stimulation
2
Macrophage Differentiation and Adipose Interaction
3
Macrophage Differentiation and Adipose Interaction
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All steps were performed using sterile techniques. Femurs were collected in RPMI (Life Technologies, Inc.). Using a needle and syringe, marrow was flushed into RPMI containing 10% FBS (Omega Scientific, Inc.) and 5% antibacterial/antimycotics (Life Technologies, Inc.). Red blood cells were lysed using ACK lyses buffer (Quality Biological) and lysis was neutralized with complete RPMI. Bone marrow cells were differentiated into macrophages using MCSF (10ng/ml; R&D) and L929 conditioned media. Non-adherent cells were collected on day 7, counted and replated. BMDMs were treated on day 8. Cells were primed by four hour treatment with ultrapure LPS (1ug/ml;Sigma) alone; inflammasome stimulation was provided by treatment with ATP (5mM; 1hr). For co-culture with adipose, BMDMs were treated as described, media removed, cells washed and weighed VAT added to culture. VAT was stimulated with 1uM NE after 1 hour, and glycerol or FFA was measured at assay end.
4
In Vitro Expansion of Antigen-Specific PBMCs
5
PBMC Isolation and Expansion for In Vitro Studies
6
In vitro expansion and functional analysis of PBMC-derived T cells
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Culturing of PBMCs for in vitro expansion was performed by incubating in RPMI (Omega Scientific) supplemented with 5% human AB serum, GlutaMAX (Gibco), and penicillin/streptomycin (Omega Scientific) at 2 × 106 per mL in the presence of BP lysates at 10 μg/mL. Every 3 days, 10 U/mL IL-2 in RPMI medium was added to the cultures. After 14 days in culture with the BP lysate, the expanded T cells were tested for the recognition of the epitope pools or individual peptides as described above. As readout, the standard IFNγ/IL-5 and IL-17/IL-9 cytokine combination for ELISpot assay or IFNγ/IL-5/IL-13 cytokine combination for FluoroSpot assay was performed as described [9 (link), 34 (link)]. For ex vivo determinations, the same combination of cytokines was used after 20 h incubation with lysate (10 μg/mL), epitope pools (2 μg/mL), or individual peptides (10 μg/mL) besides PHA (2 μg/mL) and DMSO as positive and negative controls, respectively. Consistent with these previous studies in order to be considered positive, a response in both in vitro or ex vivo modalities had to match all three different criteria: (1) eliciting at least 20 spot-forming cells (SFC) per 106 PBMCs; (2) p ≤ 0.05 by Student's t-test or by the Poisson distribution test; and (3) stimulation index (SI) ≥ 2.
7
PBMC Isolation and Expansion for In Vitro Studies
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Venous blood was collected in heparin-containing blood bags or tubes. Peripheral blood mononuclear cells (PBMC) were purified from whole blood by density-gradient centrifugation, according to the manufacturer’s instructions. Cells were cryopreserved in liquid nitrogen suspended in FBS containing 10% (vol/vol) DMSO. Culturing of PBMCs for in vitro expansion was performed by incubating in RPMI (Omega Scientific) supplemented with 5% human AB serum (Gemini Bioscience), GlutaMAX (Gibco), and penicillin/streptomycin (Omega Scientific) at 2 × 106 per mL in the presence of individual peptide pools at 5 μg/ml. Every 3 days, 10U/ml IL-2 in media were added to the cultures.
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