(Figure 1 ) consisted of cutting off the distal swab tip (~0.5 cm) with clean scissors immediately after collection, placing the tip into a cryovial with 350μL of RLT Plus® (Qiagen) with 350 μL 2-beta mercaptoethanol (Invitrogen), and vortexing the cryovial for 30 seconds, and transporting the sample to the laboratory on ice for storage at −80° C. The proximal part of the swab was placed in ESwab™ liquid Amies and processed as previously described.
2 beta mercaptoethanol
Manufactured by Thermo Fisher Scientific
Sourced in United States
2-beta-mercaptoethanol is a reducing agent commonly used in biochemical applications. It helps maintain the reduced state of proteins and other biomolecules by preventing oxidation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penn strep, by Corning (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Eswab liquid amies, by Thermo Fisher Scientific (2 mentions)
Rlt plus, by Qiagen (2 mentions)
Fluorobrite dmem media, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Corning (2 mentions)
Mt10013cv, by Corning (2 mentions)
Esgro, by Merck Group (2 mentions)
Embryomax, by Merck Group (2 mentions)
Tissue culture treated polystyrene dishes, by Corning (2 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Plasmotest, by InvivoGen (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Rpmi glutamax, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
11 protocols using 2 beta mercaptoethanol
1
Sample Collection and Processing
2
Sample Collection and Processing
3
Culturing cell lines for CD19 expression
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HEK 293-FT cells from ATCC (PTA-5077) were cultured in DMEM with 10% fetal bovine serum, antibiotics and L-glutamine. HEK 293-FT cells expressing CD19 were cultured in the same medium with 100 µg/ml zeocin added. Pre-B cell line (18.81) was obtained from Dr. M. Wabl (University of California San Francisco, San Francisco, CA), and cultures in RPMI 1640 media (GibcoTM) with heat inactivated 10% fetal bovine serum, antibiotics (100U/ml penicillin and 100 microg/ml streptomycin, GibcoTM), L-glutamine and 2-beta-mercaptoethanol (5.5 microM, GibcoTM). The 18.81 B cells express CD19 on the surface, but lack surface expression of Ig receptor. !8.81 cell identity was checked by PCR of the characteristic VHDJH gene rearrangement.
4
Immortalized Mouse Embryo Fibroblasts for DNA Repair
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Isogenic SV-40 large T antigen immortalized mouse embryo fibroblasts (MEFs) that are wild type (WT), Ku70−/−, Brca2 deficient (Brca2m/m), Polq−/−, and a subclone of Polq−/-complemented by expression of human POLQ (PolqhPOLQ) were previously described (6 (link),23 (link)). MEFs expressing DR-GFP reporter were a gift from Dr. Jeremy Stark lab (24 (link)). All MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM, Corning), with 10% Bovine Calf Serum (Hyclone BCS) and 2 mM l -glutamine (ThermoFisher), and PolqhPOLQ cells were supplemented with 2 μg/ml puromycin. Mouse ES cells (TC1) were cultured with DMEM supplemented with 15% FBS (Gibco, 1972526), 10 mM HEPES pH 7.5, 10 mM NEAA, 0.1% 2-betamercaptoethanol (Gibco, 21985023), 1% Penn-Strep (Corning, 30-002-CI) and 1:500 Leukemia inhibitory factor (LIF) treated media produced from LIF-1Cα (COS) cells. All cells were maintained at 37°C in an atmosphere of 5% CO2. Cells in culture were routinely monitored for mycoplasma contamination using the Plasmo Test™ (Invivogen).
5
Immortalized OCT4+ Mouse Embryonic Fibroblasts
6
Doxycycline-Inducible MYC Cell Lines
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All three transduced cell lines (WT MYC, T58A MYC and T58I MYC) were cultured in GlutaMAX-RPMI (Gibco, NY, USA) supplemented with 10% FBS (0.01 M HEPES, 1× sodium pyruvate, 0.05 mM 2-beta-mercaptoethanol (Gibco) and 0.1 mg/mL penicillin-streptomycin (Sigma) with the addition of 2 µg/mL doxycycline. For titration experiments, each cell line was removed from doxycycline medium, washed with PBS (3×) and RPMI (3×), and subsequently cultured under 7 different concentrations of doxycycline (0, 25, 50, 100, 300, 600 and 1000 ng/mL) for 48, 96 or 144 h at 37 °C and 5% CO2.
7
Immortalized OCT4+ Mouse Embryonic Fibroblasts
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CiA:Oct4 MEF cell lines immortalized by infection of simian virus 40 large T antigen, were obtained and cultured as previously described (Hathaway et al., 2012 (link)). Briefly, cells were cultured at 37 °C 5% CO2 conditions. Base media was either FluoroBrite DMEM Media (ThermoFisher, A1896701) for imaging, or DMEM (Corning, MT10013CV) for standard cell culture. Media was supplemented with 10% FBS (Gibco, Lot:1972526), 10 mM HEPES pH 7.5, 10 mM NEAA, 0.1% 1000 × 2-betamercaptoethanol (Gibco, 21,985,023), 1% 100× Penn-Strep (Corning, 30–002-CI). Additionally, l -Glutamine (Corning, 25005CI) at 4 mM was added to FluoroBrite media.
8
Isolation and Differentiation of MDMs
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Peripheral blood mononuclear cells (PBMC) were obtained by density gradient centrifugation using Lymphoprep (AXIS-SHIELD, Scotland), and analyzed by flow cytometry for phenotypic analysis. For a subset of individuals, monocytes were then purified using negative selection (Miltenyi, USA) and LD MACS magnetic columns (Miltenyi, USA). To prepare MDM, freshly isolated monocytes in RPMI-1640 medium (Gibco, Life Technologies, USA) were seeded at a density of 1.5 x 10 6 cells/well in 6-well plates (NEST, China). Monocytes were allowed to adhere 2 hours over cover slides, and then the medium was supplemented at final concentration of 10% autologous serum, 20 ng/ml of macrophage colony-stimulating factor (M-CSF, Miltenyi, USA), 2 mM L-glutamine (Sigma Aldrich, USA), 1% pyruvate (Sigma Aldrich, USA) and 0.1% 2-beta-mercaptoethanol (Gibco Life Technologies, USA). At day 3, the medium was changed, and MDMs were collected using Cell Dissociation buffer (Gibco, USA) and used for phenotypic analysis at day 6 or 7.
9
Maintenance of Mouse Embryonic Stem Cells
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The mESC line D3 was purchased from American Type Culture Collection (ATCC, USA). Cells were cultured on gelatine-coated flasks with high-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL, Grand Island, NY, http://www.invitrogen.com) supplemented with 15 % fetal bovine serum FBS (Gibco), 1 % non-essential amino acids (Gibco/BRL), 0.1 mM 2-beta-mercaptoethanol (Gibco/BRL), 1 mM sodium pyruvate, 100 U/ml penicillin, and 0.1 mg/ml streptomycin, and maintained at 37 8C in an atmosphere of 95 % air, 5 % CO 2 . The undifferentiated state was maintained by 1,000 U/ml recombinant LIF (Gibco). For all experiments, mESCs between passages 10 and 20 were used.
10
Maintaining Undifferentiated Mouse ESCs
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