PBMC were cultured (2 x 106 viable cells/mL) in complete media containing Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, St. Loius, MO, USA) with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA), 23.8mM sodium bicarbonate (Fisher Scientific, Waltham, MA, USA), 7.5 mM HEPES (Amresco, Framingham, MA, USA), 170 μM Penicillin G (Tokyo Chemical Industry, Portland, OR, USA), 137 μM Streptomycin (Sigma, Burlington, MA, USA), 50 μM β-mercaptoethanol (Sigma, Burlington, MA, USA), 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), essential amino acid solution (Thermo Fisher Scientific, Waltham, MA, USA), non-essential amino acid solution (Thermo Fisher Scientific, Waltham, MA, USA), 500 ng/mL R848 (Invivogen, San Diego, CA, USA) and 5 ng/mL rIL-2 (R&D, Minneapolis, MN, USA) for 7–9 days at 37°C in 5% CO2 [63 (link), 81 (link)]. Conditioned medium supernatants were harvested and evaluated for total and rHA-specific IgG abundance by ELISA starting at a 1:5 dilution. Frequency of B cells amongst total viable PBMC was assessed by CD19 surface labeling and flow cytometry analysis.
Essential amino acid solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Essential amino acid solution is a laboratory product that contains a balanced mixture of the nine essential amino acids required for human nutrition. It is designed for use in cell culture, biochemical research, and other laboratory applications where a standardized source of essential amino acids is needed.
Automatically generated - may contain errors
Lab products found in correlation
Nonessential amino acid solution, by Thermo Fisher Scientific (5 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Sodium bicarbonate, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Hepes, by Avantor (2 mentions)
Ril 2, by R&D Systems (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Penicillin g, by Tokyo Chemical Industry (2 mentions)
Its supplement, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Fatty acid free bsa, by Merck Group (2 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Selenium, by Merck Group (1 mentions)
5 protocols using essential amino acid solution
1
In Vitro PBMC Stimulation Assay
2
Activation of Human PBMCs for IgG Production
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PBMCs were plated on 12-well cell culture plate (2 × 106 viable cells/mL) with complete media containing RPMI 1640 medium (MilliporeSigma) with 10% FBS (Atlanta Biologicals), 23.8 mM sodium bicarbonate (Thermo Fisher Scientific), 7.5 mM HEPES (Amresco), 170 μM Penicillin G (Tokyo Chemical Industry), 137 μM Streptomycin (MilliporeSigma), 50 μM 2-mercaptoethanol (MilliporeSigma), 1 mM sodium pyruvate (Thermo Fisher Scientific), essential amino acid solution (Thermo Fisher Scientific), nonessential amino acid solution (Thermo Fisher Scientific), 500 ng/mL R848 (Invivogen), and 5 ng/mL rIL-2 (R&D Systems) for 7–9 days at 37°C in 5% CO2 [6 (link)]. Conditioned medium supernatants were harvested and evaluated for total and rHA-specific IgG abundance by ELISA starting at a 1:5 dilution.
3
In vitro Embryo Culture and Evaluation
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Presumptive zygotes 6 h post-insemination (hpi) were washed three times with modified synthetic oviduct fluid [33 (link)] supplemented with 20 µl/ml of
essential amino acid solution (50 ×; GibcoTM), 10 µl/ml of non-essential amino acid solution (100 ×; GibcoTM), and 5% FBS, and cultured in 250-µl microdrops of the same
medium at 39.0°C in 5% CO2, 5% O2, and 90% N2 atmosphere (20–30 zygotes per microdrop under mineral oil). Day 0 was defined as the day of the IVF/ICSI. The
number of zygotes cleaving was recorded on Day 2 (48 hpi), and the number of zygotes developing to the blastocyst stage was recorded on Day 7 (168 hpi) and Day 8 (192 hpi). Blastocysts were
qualitatively analyzed based on developmental kinetics (% Day 7 blastocysts per total harvest) and developmental stage (unexpanded, expanding, or hatched blastocysts on Day 8).
essential amino acid solution (50 ×; GibcoTM), 10 µl/ml of non-essential amino acid solution (100 ×; GibcoTM), and 5% FBS, and cultured in 250-µl microdrops of the same
medium at 39.0°C in 5% CO2, 5% O2, and 90% N2 atmosphere (20–30 zygotes per microdrop under mineral oil). Day 0 was defined as the day of the IVF/ICSI. The
number of zygotes cleaving was recorded on Day 2 (48 hpi), and the number of zygotes developing to the blastocyst stage was recorded on Day 7 (168 hpi) and Day 8 (192 hpi). Blastocysts were
qualitatively analyzed based on developmental kinetics (% Day 7 blastocysts per total harvest) and developmental stage (unexpanded, expanding, or hatched blastocysts on Day 8).
4
Optimized In Vitro Embryo Culture
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The culture medium was modified synthetic oviduct fluid [28 (link), 29 (link)] supplemented with 20 µl/ml essential amino
acid solution (50 × Gibco BRL), 10 µl/ml non-essential amino acid solution (100 × Gibco BRL), 1 mM glycine, 2 mM taurine, ITS supplement (final concentrations of 5 µg/ml insulin, 5 µg/ml
transferrin, and 5 ng/ml selenium; Sigma-Aldrich), and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). Sperm-injected oocytes were cultured in groups of 10–15 in 50 µl drops of modified
synthetic oviduct fluid medium at 38.5°C in a 5% CO2, 5% O2, and 90% N2 atmosphere. Cleavage rates and blastocyst formation rates were assessed at 72 h and
192 h after ICSI.
acid solution (50 × Gibco BRL), 10 µl/ml non-essential amino acid solution (100 × Gibco BRL), 1 mM glycine, 2 mM taurine, ITS supplement (final concentrations of 5 µg/ml insulin, 5 µg/ml
transferrin, and 5 ng/ml selenium; Sigma-Aldrich), and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). Sperm-injected oocytes were cultured in groups of 10–15 in 50 µl drops of modified
synthetic oviduct fluid medium at 38.5°C in a 5% CO2, 5% O2, and 90% N2 atmosphere. Cleavage rates and blastocyst formation rates were assessed at 72 h and
192 h after ICSI.
5
Bovine In Vitro Embryo Culture
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The culture medium was modified synthetic oviduct fluid (mSOF) [24 (link),
25 (link)] supplemented with 20 μl/ml essential amino acid solution (50 ×,
Gibco BRL), 10 μl/ml nonessential amino acid solution (100 ×, Gibco BRL), 1 mM glycine, 2 mM taurine, ITS
supplement (final concentrations of 5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml selenium; Sigma-Aldrich)
and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). ICSI and IVF oocytes were cultured in groups of 10–15 in
50-μl drops of mSOF medium at 38.5 C in an atmosphere containing 5% CO2, 5% O2 and 90%
N2. Cleavage rates and blastocyst formation rates were assessed at 72 h and 192 h, respectively,
after both ICSI and IVF.
25 (link)] supplemented with 20 μl/ml essential amino acid solution (50 ×,
Gibco BRL), 10 μl/ml nonessential amino acid solution (100 ×, Gibco BRL), 1 mM glycine, 2 mM taurine, ITS
supplement (final concentrations of 5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml selenium; Sigma-Aldrich)
and 6 mg/ml fatty acid-free BSA (Sigma-Aldrich). ICSI and IVF oocytes were cultured in groups of 10–15 in
50-μl drops of mSOF medium at 38.5 C in an atmosphere containing 5% CO2, 5% O2 and 90%
N2. Cleavage rates and blastocyst formation rates were assessed at 72 h and 192 h, respectively,
after both ICSI and IVF.
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