Cells (2 × 105) were pretreated with lactate for 3 h, then treated with lactic acid and etoposide for 36 h. The comet assay was performed using the reagents from the OxiSelect Comet assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Comets were viewed using BioTek citation 5 (BioTek, Winooski, VT, USA) in the fluorescein GFP channel using a 20x and 4x objective. The DNA% in Comet Tail were measured using Software Gen5 (BioTek, Winooski, VT, USA).
Biotek citation 5
Manufactured by Agilent Technologies
Sourced in United States
The BioTek Citation 5 is a high-performance microplate reader designed for various life science applications. It offers precise measurements of absorbance, fluorescence, and luminescence in microplates. The instrument provides accurate and reliable data to support research and testing needs.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Oxiselect comet assay kit, by Cell Biolabs (1 mentions)
Biotek cytation 5 system, by Agilent Technologies (1 mentions)
Alamarblue cell viability assay, by Thermo Fisher Scientific (1 mentions)
Spss 26, by IBM (1 mentions)
Lysosensor dnd 160, by Thermo Fisher Scientific (1 mentions)
7 protocols using biotek citation 5
1
Comet Assay for DNA Damage Assessment
2
Quantification of Thyroid Hormones in Zebrafish Larvae
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Following the exposure of embryos/larvae at 120 hpf, TH levels were measured from the homogenates of pooled fish (n = 200/replicate) using an enzyme-linked immunosorbent assay (ELISA) according to the protocol described by Yu et al. (2010) [22 (link)] with minor modifications. Briefly, 200 zebrafish larvae in a glass beaker were homogenized in 200 µL of 1× PBS using a pestle tissue grinder. The samples were then centrifuged for 10 min at 5000 × g and 4 °C. The supernatant of the samples was collected and stored at −80 °C until further measurement. The levels of hormones were quantified using ELISA kits (Cusabio Biotech, Wuhan, China) for T4 (Cat no. CSB-E08489f), T3 (Cat no. CSB-E08488f), and TSHβ (Cat no. CSB-EQ027261FI) with a BioTek Cytation 5 system (BioTek, Winooski, VT, USA) according to the manufacturer’s recommended protocol. The optical density for T4, T3, and TSHβ was determined using BioTek Cytation 5 system (BioTek, Winooski, VT, USA) set to 450 nm. The limits of detection for T4, T3, and TSHβ are reportedly 20 ng/mL, 0.5 ng/mL, and 2.5 µIU/mL, respectively. The measurements of T4, T3, and TSHβ were normalized to the protein level (mg/mL). Protein levels were analyzed using a BCA protein assay kit (Thermo Fisher Scientific, Johannesburg, South Africa) and BioTek citation 5 (BioTek, Winooski, VT, USA) system set to 562 nm.
3
Lactate-Induced Hoechst Fluorescence Analysis
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The cells were first treated with or without lactate for 6 h, then the cells were rapidly washed with PBS, and the medium was replaced with fresh medium with Hoechst dye (10 μg/ml) in the presence and absence of the inhibitor of verapamil (10 μM), the cells were further incubated for 10 min at 37 °C in the incubator. Subsequently the Hoechst medium was removed and the fluorescence intensity was quantitatively analyzed using BioTek citation 5 (BioTek, Winooski, VT).
4
Urolithin A Impacts Cell Viability
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Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Beyotime Institute of Biotechnology, Shanghai, China) assay. Cells were pretreated with urolithin A (0, 10, 20μM) for 5 h, and then the medium was aspirated. MTT assay was used to detect cell viability after 24h or 48h of continuous culture. After treatment, cells were washed with PBS and incubated with fresh medium with MTT solution (0.5mg/mL) for 4h at 37°C, the resulting formazan crystals were dissolved in DMSO and absorbance at wavelength of 570nm was taken on a plate reader using BioTek citation 5 (BioTek, Winooski, VT, USA).
5
Biocompatibility of GelMA Hydrogels
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The AlamarBlueTM cell viability assay (Thermo Fischer Scientific, Eugene, OR, USA) was conducted after 1, 5, and 7 days of incubation of the GelMA+ hydrogels with the immortalized HCEC cells. Freshly prepared and sterile (UV sterilized) GelMA+ hydrogels of both the 20% w/v and 30% w/v formulations were placed carefully onto the cells in each well and incubated at 37 °C and 5% carbon dioxide. The disc-shaped gels were washed with sterile PBS for 4 min inside the cell culture hood prior to exposure to the cells. The gels were washed four times in 5 mL of fresh sterile PBS for 1 min each time to remove any unreacted photocrosslinker. At each time point, the cell culture media was removed and then 0.5 mL of 10% v/v of the AlamarBlueTM cell viability reagent prepared with serum-free DMEM/F12 media was added to each well. The resulting solution was then incubated at 37 °C and 5% carbon dioxide for 4 h. 100 µL of the solution from each well was transferred to a new 96-well plate. The fluorescence was measured (excitation 540 nm, emission 590 nm) using the BioTek Citation 5 (BioTek, Winooski, VT, USA).
6
MTT Assay for Cell Viability
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Cell viability was determined using a 3-(4,5-dimethylthiazole-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) (Beyotime Institute of Biotechnology, Shanghai, China) assay. The range of etoposide concentrations was selected to obtain concentration for 50% of maximal effect (EC50) for treated cells. After treatment, cells were washed with PBS and incubated with fresh medium with MTT solution (0.5 mg/ml) for 4 h at 37 °C, the resulting formazan crystals were dissolved in DMSO and absorbance at wavelength of 490 nm was taken on a plate reader using BioTek citation 5 (BioTek, Winooski, VT). The measured absorbance value (OD) from the cells incubated without drugs were used for 100% survival and the inhibition rate of samples were calculated, via the formula:
The Inhibition rate = ((ODcontrol-ODexperiment)/(ODcontrol)) × 100%. We then used the inhibition rate at each point to calculate EC50 values using SPSS 26.0 software package (www.ibm.com ). For the viability of transfected cells, cells were first transfected with corresponding clones. After transfection, cells were treated with etoposide followed by MTT assay at different concentration points. Each point performed in triplicate.
The Inhibition rate = ((ODcontrol-ODexperiment)/(ODcontrol)) × 100%. We then used the inhibition rate at each point to calculate EC50 values using SPSS 26.0 software package (
7
Lysosome pH Measurement with LysoSensor
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The cells were inoculated on a 96-well plate. After growing to a suitable density, preheated (37 °C) medium containing 1 µM LysoSensorDND-160® (Thermo Fisher L7545) was added. The cells were incubated for 5 min at 37 °C, and then the medium was removed and replaced after washing the cells three times with PBS. WellScan mode was used to read the plate in BioTek Citation 5 (BioTek, Winooski, VT, USA). Fluorescence was measured using Ex360nm/Em450nm and Ex380nm/Em540nm excitation/emission wavelengths, and the lysosome pH was determined from the ratio of Em540 to Em450.
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