Placental biopsies were washed in Cytowash (consisting of DME/H-21 (Gibco), 2.5% fetal bovine serum (Hyclone), 1% glutamine plus (Atlanta Biologicals), 1% penicillin/streptomycin (Invitrogen), and 1% gentamicin (Gibco) 38 (link) and dissected into 1.0 cm2 chorionic villi explants. A single villus was placed in one well of a 24-well plate containing 0.5 ml of prewarmed cell free media [DME/H-21, 2% Nutridoma (Roche), 1% sodium pyruvate (Sigma), 1% Hepes buffer (Invitrogen), 1% glutamate plus (Atlanta Biologicals), and 1% penicillin/streptomycin (Invitrogen)] with 10% fetal bovine serum and incubated at 37°C in 5% CO2/95% for 30 minutes. Vaccine mixtures for explant exposure were then prepared such that final vaccine concentrations were: BNT162b2 and mRNA-1273 vaccine 0.1 μg/mL and 1 μg/mL, as well as control medium. Testing concentrations were based on estimates of localized circulating levels of mRNA vaccines in organs of mice vaccinated for influenza 28 (link). Explants were exposed for 0.5 or 4 hours and incubated at 37°, after which 200 ul of supernatant were collected and stored at −4°C for cytokine analysis. Exposed explants were washed in PBS and then stored at −80 °C until RNA extraction, or formalin-fixed for 24 hours at room temperature, then washed in 70% ethanol and stored at −4°C until undergoing paraffin embedding.
Glutamine plus
Manufactured by Atlanta Biologicals
Glutamine plus is a laboratory product that provides a stable source of L-glutamine, an important amino acid required for cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Merck Group (3 mentions)
Gentamicin, by Thermo Fisher Scientific (3 mentions)
Hepes buffer, by Thermo Fisher Scientific (3 mentions)
Dme h 21, by Thermo Fisher Scientific (3 mentions)
Nutridoma, by Roche (3 mentions)
Glutamate plus, by Atlanta Biologicals (2 mentions)
Cytowash, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Umuc3, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Avantor (1 mentions)
Profiler plus kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using glutamine plus
1
Placental Explants Exposure to mRNA Vaccines
2
Placental Explant Exposure to mRNA Vaccines
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Placental biopsies were washed in Cytowash (consisting of DME/H-21 (Gibco), 2.5% fetal bovine serum (HyClone), 1% glutamine plus (Atlanta Biologicals, Minneapolis, MN), 1% penicillin/streptomycin (Invitrogen), and 1% gentamicin (Gibco)42 (link) and dissected into 1.0 cm2 chorionic villi explants. A single villus was placed in one well of a 24-well plate containing 0.5 mL of prewarmed cell free media [DME/H-21, 2% Nutridoma (Roche, Basel, Switzerland), 1% sodium pyruvate (Sigma-Aldrich, St. Louis, MO), 1% HEPES buffer (Invitrogen), 1% glutamate plus (Atlanta Biologicals), and 1% penicillin/streptomycin (Invitrogen)] with 10% fetal bovine serum and incubated at 37°C in 5% CO2/95% O2 for 30 min. Vaccine mixtures for explant exposure were then prepared such that final vaccine concentrations were: BNT162b2 and mRNA-1273 vaccine 0.1 μg/mL and 1 μg/mL, as well as control medium. Testing concentrations were based on estimates of localized circulating levels of mRNA vaccines in organs of mice vaccinated for influenza.28 (link) Explants were exposed for 0.5 or 4 h and incubated at 37°C, after which 200 μl of supernatant were collected and stored at -4°C for cytokine analysis. Exposed explants were washed in PBS and then stored at −80°C until RNA extraction, or formalin-fixed for 24 h at room temperature, then washed in 70% ethanol and stored at -4°C until undergoing paraffin embedding.
3
Isolation and Culture of Cytotrophoblasts
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Cytotrophoblasts (CTBs) were isolated as previously described (Hunkapiller and Fisher, 2008 (link)). CTBs intended for proteomic analyses were cultured on precoated Matrigel (BD Biosciences) plates at a density of 104,167 cells/cm2 in medium containing DME/H-21, 2% Nutridoma (Roche), 1% sodium pyruvate (Sigma), 1% HEPES buffer (Invitrogen), 1% GlutaminePlus (Atlanta Biologicals) and 1% penicillin/streptomycin (Invitrogen). Cells were incubated at 37°C in 5% CO2/95% air. Cell culture purity was determined with cytokeratin (rat polyclonal; 1:100 (Damsky et al., 1992 (link)) and vimentin (rabbit monoclonal: 1:500) immunostaining after cytocentrifugation (800 g , 5 min); relative trophoblast purity above 85% was required for subsequent experimentation.
4
Placental Tissue Collection Protocol
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All methods were approved by the UCSF Institutional Review Board. Informed consent was obtained from all donors. Second trimester placentas were collected following elective terminations and placed in cytowash medium [DME/H-21 (Gibco), 12.5% fetal bovine serum (Hyclone), 1% glutamine plus (Atlanta Biologicals), 1% penicillin/streptomycin (Invitrogen) and 0.1% gentamicin (Gibco)]. Tissue samples were stored on ice prior to dissection.
5
Cell Line Authentication and Culturing
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HCC lines and UMUC3 and T24 were obtained from American Type Culture Collection. UMUC3 and T24 were grown as described (Guin et al. 2014 (link)) except as noted below and were authenticated by the University of Colorado Cancer Center Protein Production Shared Resource using an Applied Biosystems Profiler Plus kit, which analyzed nine STR loci (Life Technologies, 4303326). After authentication, cells were frozen within 1–2 wk. Vials of cells were resuscitated <2 mo prior to being used in experiments in this study. All cells were cultured in DMEM (VWR Scientific) with GlutaminePlus (Atlanta Biologicals), 10% FBS (Seradigm), penicillin/streptomycin (GIBCO), glutamax (GIBCO), and sodium pyruvate (GIBCO), except HepG2 cells were cultured in EMEM (American Type Culture Collection) without sodium pyruvate.
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