The cell viability of AFB1 treatment on the primary trophoblast cells was assessed with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) test (Sigma-Aldrich/Merck, #M2128) as previously described by Storvik et al. (2011) (link). Human primary trophoblasts (5x105 cells/well) were incubated with 0.01–3 µM AFB1 for 72 h at 37 °C and then the MTT reagent was added for 4 h incubation, sodium dodecyl sulfate - N-dimethylformamide (SDS–DMF) (Sigma-Aldrich/Merck, #L3771 and #D4551, respectively) was added and the plate was maintained overnight at 37 °C. The optical density was measured using a BioTek ELx800 reader (BioTek, USA) at a wavelength of 570 nm. The results were expressed as a percentage of the control cells exposed to dimethyl sulfoxide to the same concentration used as the solvent for AFB1. The significance of the differences between exposures and respective controls was analyzed by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test.
D4551
Manufactured by Merck Group
Sourced in Germany
D4551 is a laboratory equipment product from Merck Group. It is a multi-functional device designed for use in various scientific applications. The core function of D4551 is to perform precise measurements and analysis tasks in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
L3771, by Merck Group (1 mentions)
Elx800 reader, by Agilent Technologies (1 mentions)
M2128, by Merck Group (1 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (1 mentions)
X gal, by Roche (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
M5655, by Merck Group (1 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
N dimethylformamide, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
L glutamine solution, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
6 protocols using d4551
1
Cell Viability Assessment of AFB1 on Trophoblasts
2
TLD Antiretroviral Compound Preparation
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One Acriptega tablet (Mylan Pharmaceuticals) containing 50 mg dolutegravir (DTG), and 300 mg each of lamivudine (3TC) and tenofovir disoproxil fumarate (TDF), the components of the first line TLD regimen. The pill was powdered using a pill crusher and powder resuspended in 25 mL of N,N-Dimethylformamide (DMF, Sigma D4551) and Dulbecco’s phosphate-buffered saline (DPBS, Whitehead Scientific PBS-1A) at a dilution of 1:4 (DMF:PBS) for a stock concentration of 2 mg/mL DTG and 12 mg/mL 3TC/TDF. Solution was maintained at 37 °C for 4 h until powder completely dissolved. TLD stocks were serially diluted in RPMI in a 2-fold dilution series and the diluted TLD was added to H1299-E3 cells plated in a 96-well plate (Corning CLS, 3595) at 20,000 cells per well for SARS-CoV-2 infection or to RevCEM-GFP cells in a 24-well plate (TPP, 92024) at 0.5 × 106 cells per well for HIV infection. Both cell types were incubated 1 day with TLD before infection with the respective virus.
3
X-Gal Staining Protocol Optimization
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X-gal staining solution was prepared following instructions of the staining kit (Senescence β-Galactosidase Staining Kit, #9860 Cell Signaling Technology). For each mL of X-gal Staining Solution, 930 µl of 1X staining buffer (837 µl of ddH2O + 93 µl of 10X staining buffer), 10 µl of solution A, 10 µl of solution B, and 50 µl of X-gal solution (20 mg/ml X-gal dissolved in DMF) were mixed in a 15 mL falcon tube. The optimal pH of the solution was measured to be 5.5–6.0. It is important to note here that two variables, pH and DMF played a role in determining the staining. In particular, staining was optimal when pH of the staining solution was 5.5–6.0, and X-gal should be dissolved in DMF from Sigma with the highest purity ≥ 99% (#D4551, Sigma). Also, only the X-gal provided in the kit works best for this staining protocol (we tried X-gal from Roche #10,651,745,001).
4
Oligonucleotide-HaloTag Ligand Conjugation
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Oligonucleotides to be conjugated with the HaloTag ligand were synthesized with a 5’ C12-linked amine and a 3’ biotin group (IDT). Oligonucleotides were initially ethanol-precipitated and subsequently resuspended to 1 mM in conjugation buffer (100 mM Na2HPO4 (Sigma-Aldrich S9763), 150 mM NaCl (Thermo Fisher Scientific S271), pH 8.5). Resuspended oligos were combined with an equal volume of the HaloTag ligand succinimidyl ester (O4) (Promega P6751) resuspended in N,N- dimethylformamide (Sigma-Aldrich D4551) with a 30-fold molar excess of the ligand. Conjugation reactions were conducted overnight at room temperature with constant agitation prior to final cleanup via ethanol precipitation.
5
MTT Assay for Cell Viability
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A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (M5655; Sigma-Aldrich) assay was conducted to measure the cell viability of SH-SY5Y cells. Cells were seeded in 48-well plates with growth media. When the cells reached 70% confluency, they were treated with DMSO or BTZ. After 2 days of treatment, MTT solution (1 mg/mL) was added to the media and left to incubate for 2 h at 37°C to form formazan crystals. Lysis buffer composed of 20% SDS [0227; VWR (Amresco)] in 50% aqueous N, N-dimethylformamide (D4551; Sigma-Aldrich) was added to solubilize formazan for 30 min. Lysates were transferred to 96-well plates, and absorbance was estimated at 550 nm by a Synergy H1 Hybrid Multi-Mode microplate reader (Biotek, Winooski, VT, USA).
6
Murine Bone Marrow-Derived Dendritic Cell Culture
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Bone marrow cells were harvested from tibiae and femurs of mice, erythrocytes were lysed with ACK lysis buffer, filtered and cultured in VLE-DMEM media (FG1445; Merck Millipore) supplemented with 5% culture supernatants containing murine GM-CSF (prepared in house from X63-GM-CSF cell line culture), or 20% of supernatant containing murine FLT3L (prepared in house from CHO FLT3-L FLAG cell culture; CHO FLT3-L FLAG cell line was kindly provided by Tim Sparwasser Institute of Infection Immunology, TWINCORE, Hannover, Germany), heat inactivated 10% fetal calf serum (10270; Gibco), 1% of penicillin, streptomycin, L-glutamine solution (G6784 Sigma), and 0.1% 50mM beta mercaptoethanol (D4551, Sigma), which constitute a complete media (CM) and cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Bone marrow cells seeded with 3 × 106 cells per petri dish (58 cm2) in 10 ml of CM or in 24-well plates with 2 × 105 cells/well in 1 ml CM. On day 3, extra CM was added with volume equal to initial volume. On days 5–6, cells were either harvested for seeding in 24 well plates or directly treated in petri dishes.
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