Cell viability was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega #G9242) according to manufacturer’s instructions. Briefly, 5,000 cells were seeded (in triplicate/condition) in a solid bottom white-walled 96-well plate and incubated with different concentrations of the drug for 5 d. Luminescence signal was detected using a BioTek Synergy HTX Multi-Mode microplate reader (Agilent company), and relative light unit (RLU) was analyzed with GraphPad Prism 8.4.3 (GraphPad software). Percentage of cell viability was calculated by dividing the RLU of drug-treated sample by the RLU of the control (drug vehicle) and multiplying by 100. Another method of cell viability analysis was by mixing cells with Trypan Blue Solution, 0.4% (Thermo Fisher Scientific #15250061), in a 1:1 dilution and inserting into a cell-counting chamber slide (Thermo Fisher Scientific #C10312) to read in a Countess™ 3 FL Automated Cell Counter (Thermo Fisher Scientific #AMQAF2000).
Countess 3 fl automated cell counter
Manufactured by Thermo Fisher Scientific
Sourced in Spain
The Countess 3 FL Automated Cell Counter is a laboratory instrument designed to count and analyze cells. It provides automated cell counting and viability assessment using fluorescence microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Prism 8, by GraphPad (1 mentions)
Synergy htx multi mode microplate reader, by Agilent Technologies (1 mentions)
Celltiter glo 2.0 cell viability assay, by Promega (1 mentions)
Block it alexa fluor red fluorescent control, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Trypan blue stain, by Thermo Fisher Scientific (1 mentions)
C6295, by Merck Group (1 mentions)
Evos m5000 fluorescence inverted microscope, by Thermo Fisher Scientific (1 mentions)
Tib 67, by American Type Culture Collection (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
11 protocols using countess 3 fl automated cell counter
1
Cell Viability Assessment Protocols
2
Silencing Purinergic and Toll-like Receptors
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MMG were transfected with Ambion Silencer Select P2RX7 (ID no. s9959), TLR2 (ID no. s76898), TLR4 (ID no. s14195), TLR7 (ID no. s27842), TLR8 (ID no. s27920), or control (catalogue no. 4390846) siRNA (siNS) using lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Gibco) according to the manufacturer’s instructions. Cells were analysed for target gene silencing 48 h post-transfection and used in experiments. Transfection efficiency was assessed using BLOCK-iT Alexa Fluor red fluorescent control (Invitrogen) on a Countess 3 FL automated cell counter (Thermo Fisher).
3
Culturing Mouse Proximal Tubule Cells
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BUMPT (Boston University Mouse Proximal Tubule clone 306), a mouse proximal tubule cell line33 (link), 48 (link) was maintained in DMEM with L-glutamine (Thermo) supplemented with 5% fetal bovine serum (Corning) and 100 U/ml of penicillin/streptomycin (Thermo). BUMPT cells were passaged in humidified 5% CO2 at 37 °C, at sub-confluence. Mycoplasma contamination of parental BUMPT cells used in this study was tested with LookOut mycoplasma PCR detection kit (Sigma). To collect the conditioned media, BUMPT cells were cultured for the indicated times, using serum-free medium, supplemented with L-glutamine and 100 U/ml of penicillin/streptomycin. For cell culture experiments, equal numbers of viable cells were plated after cell numbers were counted twice or three times, using Countess 3 FL Automated Cell Counter (Thermo) with Invitrogen viability test kit (Thermo).
4
HEK293T Cell Maintenance and Transfection
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HEK293T cells were cultured using DMEM (Gibco) supplemented with 10% FBS (Capricorn Scientific) and 1% Penicillin–Streptomycin (ThermoFisher) in a 12-well format. When reaching a confluency of 90% the cells were split. Each well was washed once with PBS and 100 μl of Trypsin (Gibco) was added. After incubation for 3 min at 37°C the detached cells were collected in a 15 ml tube. Cells were counted with the Countess 3 FL Automated Cell Counter (ThermoFisher) and seeded at a density of 75.000 cells/well in 1 ml medium for maintenance. For transfection the cells were seeded at a density of 350.000 cells/well in 1 ml medium.
5
Immortalized Cell Culture Cryopreservation and Passage
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Immortalized cells were cultured according to standard practices. Briefly, cells were cryopreserved in low glucose DMEM (11885084, Thermo Fisher, Darmstadt, Germany) supplemented with 20% FBS (S0615-500ML, Sigma-Aldrich, Taufkirchen, Germany) and 10% DMSO (C6295, Sigma-Aldrich) in liquid N2. To bring them in culture, cryovials were thawed and resuspended in warmed complete medium made of DMEM supplemented with 10% FBS and 1% Anti/Anti (15240062, Thermo Fisher). The cell suspension was centrifuged at 0.5 rcf for 5′ to remove the DMSO, the cell pellet was resuspended in complete medium and transferred to a sterile T-75 flask. Cells were maintained in an incubator at 37 °C, 5% CO2, 85–95% humidity and passaged once 90% confluent. For passaging, the medium was aspirated, the cells washed once with DPBS-CaCl2 and then detached from the flask using 0.05% Trypsin-EDTA (5′ incubation at 37 °C, 5% CO2). Cells were counted using 0.4% Trypan Blue Stain (T10282, Thermo Fisher) and the Countess3 FL Automated Cell Counter (Thermo Fisher) before seeding in a new recipient.
6
Programmed Cell Death in N. fowleri
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A selection of the products of relevance, those with the highest selectivity index, was used to study programmed cell death (PCD) in N. fowleri (ATCC® 30,808™). Therefore, the trophozoites were incubated in a 96-well plate at a concentration of 5 × 105 cells/mL making use of a Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific, Spain). Once attached to the wells, they were treated with IC90 of the chemicals during 24 h. In order to expose the different metabolic events characteristic of a PCD, the reagents were added according to manufacturer's instructions under dark conditions to avoid their degradation. The results were revealed by fluorescence obtained using EVOS™ M5000 fluorescence inverted microscope (Invitrogen, Thermo Fisher Scientific, Madrid, Spain). For each objective lens thickness (40 × and 100 × ), five images were taken. The percentage of stained cells after the incubation of the cells with the dyes was also calculated. Moreover, a one-way analysis of variance (ANOVA) was used for data analysis on order to evaluate the differences between negative control and treated cells. The results are shown as the mean value of three different images ± standard deviation (SD).
7
Cytotoxicity Evaluation of Compounds
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For the evaluation of cytotoxicity of the products of interest, a murine macrophage cell line J774A.1 (ATCC® TIB-67™) was used. The macrophages were cultured in 96-well plates at a concentration of 105 cells/mL using the Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific, Spain) with RPMI (Roswell Park Memorial Institute, 1640) medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Subsequently, the diluted compounds were added at different concentrations. Finally, 10% of the total volume of alamarBlue® was added as previously mentioned. The plate was read using an EnSpire® Multimode Plate Reader (PerkinElmer, Madrid, Spain) and analyzed in GraphPad Prism 9.0 to determine the cytotoxic concentration (CC50) and subsequently obtain the selectivity index (CC50/IC50).
8
Long-term Expansion of Engineered T Cells
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A total of 2 × 106 T cells were cultured in 2 mL R10 media in 12-well plates and replated in fresh R10 media every 3 to 4 d for 28 d. At each medium change, cells were maintained at a concentration of 1 × 106 cells/mL and counted using the Countess 3 FL Automated Cell Counter (Invitrogen) using Trypan blue (Gibco) as a viability stain. NPM–ALK-transduced T cells were used as a positive control for transformed T cells and have been described previously (34 (link), 35 (link)).
9
Single Cell Sequencing Protocol
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Single cell samples were assessed for proper quality control metrics prior to library preparation. All samples were quantitated through the Invitrogen Countess 3 FL Automated Cell Counter (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) using ReadyCount Green/Red Viability Stain (ReadyCount Green/Red Viability Stain) per manufactures’ protocol with GFP filter. Samples were all greater than 90% viable with cell density averaging 1000 cells/ul. A total of 10000 cells per sample were used for downstream library preparation. Single cell samples were processed through the 10X Genomics Chromium Single Cell 3’ protocol v3.1 per manufactures’ instruction. Single cell droplets (GEMs) were produced using the 10X Genomics Chromium Controller and barcoded for sample identification. Final libraries were validated on the Qiagen QIAxcel DNA High Sensitivity Bioanalyzer (QIAGEN, Germantown, MD, USA)), to visualize proper insert size, and concentration was calculated using the KAPA Illumina Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) on the Bio-Rad CFX96 thermal cycler (Bio-Rad Inc., Hercules, CA, USA). Pooled libraries were sequenced on the Illumina NextSeq 2000 per 10X Genomics specifications (28x91 cycles).
10
Cell Preparation and Perturbation Workflow
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HEK-293T cells were cultured at 37 C and 5% CO2 in Dulbecco’s Modified Eagle’s High Glucose media supplemented with 10% Fetal Bovine Serum. Cells were detached with extensive phosphate-buffered saline (PBS) resuspension, cells were pelleted and washed for a total of 3 times. Cells were counted using the Countess™ 3 FL Automated Cell Counter (SN: AMQAF2000, Invitrogen) and strained using a 5-mL cell strainer tube (SN: 352235, Falcon®) to a final concentration of 200 cells/μL for optimal cell dispensing on the cellenONE®. For the proteome perturbation experiment, HEK-293T cells were seeded in a 6-well plate (Costar® 6-well plates, 3506, Corning Incorporated) at a density of 3 × 105 cells/well. THP-1 cells were seeded in a T25 flask at a density of 4 × 105 cells/mL in Gibco RPMI media supplemented with 10% Fetal Bovine Serum, Penicillin-Streptomycin and Glutamax. After 8-hours, media was supplemented with LPS (final conc. of 200 ng/mL in DMSO) or DMSO-control. Treatment was carried out for 12-hours prior to cell pelleting, washing, and counting. This was followed by cell straining and subsequent sample processing within the cellenONE.
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