Venous blood was collected in 2 heparinized tubes (BD Vacutainer) of 10 ml each, centrifuged at 1,500 rpm for 7 min and the plasma was taken and kept at −80°C. The rest of the blood was diluted with HBSS medium (Gibco) supplemented with Pen-Strep-Glu (Gibco), delicately deposited on Ficoll (GE Heathcare) and centrifuged for 25min at 1500rpm. The mononuclear cell layer was collected in supplemented HBSS medium and washed twice. Cells were then suspended in complete medium (RPMI 1640 without L-Glutamine (Gibco) supplemented with 1% Pen Strep Glu (Gibco), 1% Sodium pyruvate (Gibco), 10% Fetal Bovine Serum (Sigma-Aldrich)), counted under the microscope using Turk solution (Merck), before being suspended in a freezing medium (RPMI 1640 without L-Glutamine supplemented with 1% Pen Strep Glu (Gibco), 1% Sodium pyruvate (Gibco), 20% Fetal Bovine Serum (Gibco), 10% DMSO (Merck)) at a concentration of 107 cells per ml and frozen at −80°C for 24h before being transferred to −150°C.
Rpmi 1640 without l glutamine
Manufactured by Thermo Fisher Scientific
Sourced in United States
RPMI 1640 without L-glutamine is a cell culture medium commonly used for the in vitro cultivation of a variety of cell types, including mammalian cells. It is a formulation of various salts, amino acids, vitamins, and other components essential for cell growth and maintenance, but does not include the amino acid L-glutamine.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Albumin fraction 5, by Merck Group (1 mentions)
Activin a, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Sk n sh, by American Type Culture Collection (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Lymphoprep medium, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Bovogen (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
7 protocols using rpmi 1640 without l glutamine
1
Freezing and storing human PBMCs
2
Hepatic Differentiation of Human PSCs
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For the hepatic differentiation of human PSCs, a previously reported hepatic differentiation protocol designed for human ESCs [1 (link)] was applied with some modifications. Briefly, human PSCs were plated at a density of 2.5 × 105 cells/ml on Matrigel-coated six-well plates with mTeSR1 medium (STEMCELL Technologies) including ROCK inhibitor (Y27632; STEMCELL Technologies). The medium was replaced with definitive endodermal induction medium (DE) for 5 days (stage I). The DE medium consisted of RPMI 1640 (without l -glutamine; Gibco-BRL) supplemented with 2 mM l -glutamine (Millipore), 0.5 mg/ml albumin fraction V (Merck Millipore, Germany) and 100 ng/ml Activin A (PeproTech, NJ, USA). Afterward, the definitive endodermal cells were differentiated into hepatoblasts using the hepatic endodermal medium (Hep-1) for 5 days (stage II) followed by hepatic specification medium (Hep-2) for 5 days (stage III). Hep-1 and Hep-2 comprised the HBM SingleQuots™ kit (Lonza) supplemented with 30 ng/ml FGF4 (PeproTech) and 20 ng/ml BMP2 (Invitrogen, MD, USA) at stage II and 20 ng/ml HGF (PeproTech) and 20 ng/ml OSM (R&D Systems, MN, USA) at stage III, respectively. After hepatic specification, the cells were further matured using the HMM SingleQuots™ kit for 5 days (stage IV).
3
Cell line growth and differentiation
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The following cell lines and media for routine growth of these cell lines were used: C20 (a kind gift from Dr. Alvarez-Carbonell [25 (link)])—DMEM with 4.5 mg/mL glucose, no pyruvate (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 5% fetal bovine serum (FBS), antibiotics; HMC3 and SK-N-SH (American Type Culture Collection [ATCC])—EMEM with 2 mM L-glutamine, 1 mM sodium pyruvate (ATCC), 10% FBS, and antibiotics; A172 and THP-1 (ATCC)—RPMI-1640 without L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS, 2 mM L-alanyl-L-glutamine dipeptide (GlutaMAX; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), antibiotics; BHK-ABCA1 (a kind gift from Dr. Chongren Tang [26 (link)]) and BHK-ABCA7 [10 (link)]—DMEM with 4.5 mg/mL glucose, no pyruvate (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS, and antibiotics. All cells were grown at 37 °C in 5% CO2. For differentiation into macrophages, THP-1 cells were treated with 10 ng/mL PMA (P8139; Sigma-Aldrich, St. Louis, MO, USA) for 24 h.
4
Cytokine Response to Hookworm Peptides
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The human blood for this research was donated by healthy volunteers. Donor material was studied under the guidelines and regulations of James Cook University (JCU; Cairns, Australia; H7010). Written informed consent was obtained from all participants of the study. PBMCs were isolated from whole blood by density gradient centrifugation using Lymphoprep™ medium (STEMCELL™ Technologies, Vancouver, Canada), according to the manufacturer’s instructions. During experiments, cells were maintained in a culture media containing RPMI 1640 without L-glutamine (Gibco Thermo Fisher Scientific, Waltham, MA, United States), 10% heat-inactivated fetal bovine serum (FBS) (Bovogen Biologicals, Christchurch, NZ), 10,000 units/mL of penicillin + 10,000 μg/mL of streptomycin (Thermo Fisher Scientific), and 1X GlutaMAX (Thermo Fisher Scientific).
The induction of cytokine secretion in vitro was achieved by using either a cell stimulation cocktail of 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (eBioscience), or 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich). Under each condition, cells were treated with 0.1–100 μg/mL of hookworm AIP peptides or remained untreated. The cell culture plates were incubated for 24 h at 37°C and 5% CO2. After incubation, the samples were centrifuged at 500 × g for 5 min, and the culture supernatants were collected for cytokine analysis.
The induction of cytokine secretion in vitro was achieved by using either a cell stimulation cocktail of 50 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 1 μg/mL ionomycin (eBioscience), or 10 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich). Under each condition, cells were treated with 0.1–100 μg/mL of hookworm AIP peptides or remained untreated. The cell culture plates were incubated for 24 h at 37°C and 5% CO2. After incubation, the samples were centrifuged at 500 × g for 5 min, and the culture supernatants were collected for cytokine analysis.
5
Monocyte-Derived Macrophage Generation
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To generate MDMs, MACS-isolated monocytes were seeded in 24-well plates at 5x105 cells/well and cultured (at 37°C, 5% CO2) for 7 days in RPMI 1640 without L-glutamine (Gibco, #11875085), supplemented with 10% HI-FCS (Scientifix life, FFBS-500), 100 units(μg)/mL penicillin/streptomycin (Gibco, #15140122), MEM-NEAA (Gibco, #11140050), Glutamax (Gibco, #35050061), 100 ng/mL macrophage colony-stimulating factor (M-CSF) (Peprotech, #300-25), and/or 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, #300-03). GM-CSF was used only for experiments with monocytes isolated from healthy donors. Media were not changed for the duration of the experiments.
6
Cryopreservation of PBMCs from Whole Blood
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For each donor, 4 mL cryopreservation medium was prepared, consisting of 3.2 mL RPMI 1640 without L-Glutamine (ThermoFisher Scientific Inc., Waltham, USA) and 0.8 mL DMSO (OriGen Biomedical, Austin, USA). Mixing was performed by dropwise addition of the 4°C cryopreservation medium to the PBMCs suspended in HSP. Thus, the final concentration of DMSO was 10%. For each donor, 1 mL cell suspension was added to each of 8 cryotubes, with each tube thus containing a number of PMBCs corresponding to that obtained from 1 mL whole blood. The cryotubes were then immediately placed directly in a rack in a -80°C freezer, where they were stored until thawing. Samples were stored for up to 2 weeks before thawing and cell counting.
7
Purification and Culture of Mouse B Cells
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All in vitro culture experiments were performed with B cells purified by negative selection. B cell purification was generally performed as described (Sullivan et al., 2011 (link)), but with the following modifications: Media used was DMEM (Thermo Fisher Scientific) containing 4.5 g/liter of glucose, 2% FBS (Life Technologies), and 10 mM Hepes (Life Technologies). Centrifugation steps were performed at 400 g for 5 min. ACK lysis was not performed. αCD11c biotin (clone N418; Biolegend) was used at 1 μl per 108 cells, and αTER-119 biotin (clone TER-119; Biolegend) was used at 10 μl per 108 cells. 100 μl of Dynabeads MyOne Streptavidin T1 (Life Technologies) were used per 108 cells. In the case of Igλ B cell enrichments, 10 μl of αIgκ biotin (clone RMK-12; Biolegend) were added per 108 cells and 200 μl of Dynabeads MyOne Streptavidin T1 were used per 108 cells. After purification, cells were resuspended in complete RPMI (cRPMI), composed of RPMI 1640 without L-glutamine (Thermo Fisher Scientific), 10% FBS, 10 mM Hepes, 1X penicillin streptomycin L-glutamine (Thermo Fisher Scientific), and 50 μM β-mercaptoethanol (Thermo Fisher Scientific). Final purity was assessed by flow cytometry. Total B cell purity routinely exceeded 98%. For Igλ-enriched purifications ∼80% of B cells obtained were routinely Igλ+.
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