Human macrophages were obtained as described by Gray et al. [33 (link)]. Briefly, human peripheral blood (New York Blood Center, NY, NY) was fractionated utilizing density gradient centrifugation (Ficoll-Paque Premium, 1.077 g/mL, GE Healthcare, Chicago, IL). The buffy coat was collected and washed with 1× PBS and the mononuclear cells isolated by enriching the CD14+ cell population, using magnetic bead cell sorting according to manufacturer's instructions (Miltenyi Biotech, Charleston, MA). Monocytes were seeded at 1 × 107 cells/T-175 cm2 flasks in Advanced RPMI supplemented with 10 % FBS, 1 % P/S, 4 mM l -glutamine, and 5 ng/mL granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) to induce differentiation into proinflammatory M1 macrophages. After 7 days, differentiated cells were detached with trypsin-EDTA and cryopreserved at passage 1 in fully supplemented Advanced RPMI (ThermoFisher, Boston, MA) containing 10 % dimethyl sulfoxide. Passage 1 macrophages were used for our studies by quickly thawing and culturing them in fully supplemented Advanced RPMI overnight to allow cell attachment.
L glutamine
Manufactured by R&D Systems
Sourced in United States
L-glutamine is a naturally occurring amino acid that plays a vital role in various cellular processes. It is an essential component for cell growth and maintenance. L-glutamine serves as a building block for proteins and is involved in the synthesis of other amino acids, nucleic acids, and important biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by R&D Systems (6 mentions)
Penicillin, by R&D Systems (5 mentions)
Epidermal growth factor (egf), by R&D Systems (5 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Gm csf, by R&D Systems (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Interleukin 7 (il 7), by R&D Systems (3 mentions)
Matrigel, by BD (3 mentions)
Penicillin streptomycin, by R&D Systems (3 mentions)
Hepes, by R&D Systems (3 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (3 mentions)
Rpmi 1640, by R&D Systems (3 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions)
Activin a, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
30 protocols using l glutamine
1
Isolation and Differentiation of Human M1 Macrophages
2
Plasmacytoid Dendritic Cell Cytokine Regulation
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Isolated pDCs (5 × 104 cells/96-well cavity) were seeded in 200 µl of RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 100 IU ml−1 penicillin, 100 µg ml−1 streptomycin, 2 mM l -glutamine, 10% (vol/vol) human serum and recombinant human (rh) IL-3 [10 ng ml−1; R&D Systems]. To determine the levels of secreted IFNα, pDCs were cultured for 12–14 h in the presence of ODN 2216 (CpG A; 0.26 µM; Thermo Fisher Scientific). To block p75NTR activity, the inhibitory peptide TAT-Pep5 (100 nM, Merck) was added. Recombinant human β-NGF (R&D Systems) was added at the indicated concentrations. The IFNα levels in supernatants were determined using an ELISA (eBioscience). To determine the levels of secreted IL-6, pDCs were stimulated with an antibody specific to human FcεRlα (250 ng ml−1; eBioscience) alone or in the presence of the TAT-Pep5 inhibitor. Recombinant human β-NGF was added at the indicated concentrations. After 14 h, the supernatant was collected to measure IL-6 levels using an ELISA (eBioscience).
3
Efficient hPGCLC Induction from hESCs
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Induction of hPGCLCs was performed according to a previous report [26 (link)]. Briefly, hESCs were dissociated with 0.5 mM EDTA/PBS and 1 × 106 cells per well were plated on Matrigel coated 6-well plates in GK15 medium (G-MEM [Thermo Fisher, 11,710–035], 15% KSR [Thermo Fisher, 10,828–028], 0.1 mM NEAA [Thermo Fisher, 11,140–050], 2 mM L-glutamine [Thermo Fisher, 35,050–061], 1 mM sodium pyruvate [Thermo Fisher, 11,360–070], 0.1 mM 2-mercaptoethanol [Sigma, M3148], 3 μM CHIR99021 [Selleck Chemicals, S2745], 50 ng/ml Activin A [PEPRO TECH, 120-14E] and 10 μM ROCK inhibitor) for pre-induction. After 40 ~ 42 h of pre-induction, the cells were dissociated with Accutase (Thermo Fisher, A1110501) and plated into ultra-low cell attachment U-bottom 96-well plates (Corning, 7007) at a density of 2,000–4,000 cells per well to form embryoid bodies in 200 μl of aRB27 induction medium (Advanced RPMI 1640 [Thermo Fisher, 12,633–012], 1% B-27 supplement [Thermo Fisher, 17,504–044], 0.1 mM NEAA, 2 mM L-glutamine, 500 ng/ml BMP4 (R&D Systems, 314-BP-050), 10 ng/ml human LIF (R&D Systems, 7734-LF-100), 100 ng/ml SCF (R&D Systems, 255-SC-050), 50 ng/ml EGF (R&D Systems, 236-EG-200), and 10 μM ROCK inhibitor (Selleck, S1049) or 10 μM SB431542 (Selleck, S1067).
4
In Vitro T Cell Differentiation
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After 14 days of respecification, 1×105 5F were plated in OP9-DL1 stromal co-culture33 . Cells were cultured in α-MEM (Gibco), 1% penicillin/streptomycin, 20% FBS (Gemini), and 1 mM L-glutamine with 30 ng/mL SCF, 5 ng/mL FLT3, 5 ng/mL IL-7 (all R&D Systems) for 20 days with 2 μg/mL Dox followed by Dox removal. Cells were harvested by mechanical dissociation and filtered through a 40 μM filter and passaged onto fresh stroma every 5-7 days. T cell development was assessed after 35 days using CD45, CD7, CD3, CD4 and CD8.
5
In Vitro T Cell Differentiation
6
Generation and Maintenance of Mouse MLL/AF10 Leukemia Cell Lines
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Mouse MLL/AF10 leukemia cell lines have been generated by retroviral transduction of MLL/AF10(OM-LZ) and wild-type (KRASwt) or activating KRAS (KRASG12C) to 5-fluorouracil-enriched C57BL/6 (B6; National Laboratory Animal Center, Taiwan) mouse bone marrow (BM) cells in our previous studies [13 (link),23 (link),24] . Of these mouse leukemia cell lines, 12G cells harbor MLL/AF10(OM-LZ) solely, AK2G and AK3G cells harbor MLL/AF10(OM-LZ) and KRASG12C, and AKw1G cells harbor MLL/AF10(OM-LZ) and KRASwt. Mouse leukemia cell lines were maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 20% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 μM 2-mercaptoethanol, and 10 ng/ml interleukin-3 (R&D Systems, Minneapolis, MN, USA).
7
3D Culturing of Gut Organoids
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The spheroids were transferred to a three-dimensional in vitro culture system as previously described6 (link),10 (link). Briefly, spheroids were collected, resuspended in 50 μl Matrigel (BD Biosciences), and plated in a three-dimensional droplet. After Matrigel was allowed to solidify for 10–15 minutes in a tissue culture incubator, spheroids were overlaid with gut media: Advanced DMEM/F12 with N2 (Invitrogen), B27 (Invitrogen), L-glutamine, 10 μM HEPES, penicillin/streptomycin, and EGF (100 ng ml−1; R&D Systems). For the first three days, RA and Noggin were added to the gut media. Media was replaced every 3–4 days, as necessary. At day 20, organoids were collected and re-plated in fresh Matrigel at dilution of ~1:12.
8
3D Culturing of Gut Organoids
9
Rapid HPIV3-Specific VST Protocol
10
Isolation of Bone Marrow-Derived Macrophages
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Bone marrow-derived macrophages were isolated from the femur and tibia bones of 6- to 8-week-old C57BL/6 male mice using a previously described protocol [30 (link)]. BMDMs were grown in RPMI 1640 medium with 10% heat-inactivated FBS, 1% nonessential amino acids (NEAAs), 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 20 ng/mL macrophage colony-stimulating factor (R&D Systems). The animal study was approved by the Institutional Animal Care and Use Committee (IACUC, NO. 1051219) at National Yang-Ming University (Taipei, Taiwan).
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