shCTRL and shANK cells (7 × 104) were seeded in octuplicate into a 96-well plate, and allowed to adhere overnight in a humidified atmosphere of 5% CO2. Cells were treated with and without 21 μg/mL of Trastuzumab (Herceptin® 150 mg, Roche, Basel, SW) at 37°C for 48 h, incubated with WST-1 reagent (Clontech Laboratories, Mountain View, CA) for 1 h at 37°C and read at 450 nm with the Infinite® 200 PRO NanoQuant plate reader (Tecan) to assess viability. Values were then normalized on the respective untreated cells.
Infinite 200 pro nanoquant plate reader
Manufactured by Tecan
Sourced in Switzerland, Germany
The Infinite® 200 PRO NanoQuant plate reader is a microplate-based instrument designed for the quantitative analysis of nucleic acids and proteins. It features a wide measurement range, high sensitivity, and versatile data analysis capabilities.
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Lab products found in correlation
Sensolyte pnpp alkaline phosphatase detection kit, by AnaSpec (2 mentions)
Wst 1 reagent, by Takara Bio (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
Cellrox oxidative stress reagent, by Thermo Fisher Scientific (1 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
5 mc dna elisa kit, by Zymo Research (1 mentions)
10 protocols using infinite 200 pro nanoquant plate reader
1
Trastuzumab Modulates Cell Viability
2
Quantifying pNF-H in Aqueous Humor
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The aqueous humor collected from AOH and COH models was used for quantifying pNF-H through an enzyme-linked immunosorbent assay (ELISA) kit (Phosphorylated Neurofilament Sandwich ELISA Kit, Merck KGaA, Germany), and the analysis was carried out according to the manufacturer's protocols. The samples were diluted at a ratio of 1:6 with a sample diluent. pNF-H antibody-coated strip wells were incubated with 50 μl of standards or samples at room temperature (RT) for 1 h with gentle shaking. Plates were washed six times with diluted wash buffer provided in the kit; then, 100 μl of detection antibody was added to all wells and incubated for 1 h at RT. Plates were washed six times and 100 μl of the diluted alkaline phosphatase-conjugated goat anti-rabbit polyclonal antibody was added to each well and incubated for 1 h. After six washes, 100 μl of freshly diluted 1× pNPP alkaline phosphatase substrate was added to each well and incubated in the dark at RT for 30–60 min. Stop solution (3N NaOH) was added and plates were read at 405 nm on an Infinite 200 PRO NanoQuant Plate reader (Tecan, Switzerland) using i-control analytical software.
3
NF-kB p65 Transcription Factor Assay
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Proteins were extracted using Nuclear Extraction Kit (Abcam) according to manufacturer procedure and concentrations were determined by Bradford reagent (Sigma-Aldrich). NF-kβ p65 Transcription Factor Assay Kit (Abcam) was used for measure the p65-RELA transcription factor DNA binding activity. A known concentration of nuclear and cytoplasmic proteins were loaded into a 96-well containing a double stranded DNA (dsDNA) sequence highly specific for the NF-kβ response element immobilized onto the bottom of wells according to manufacturer procedure. The activity of NF-kβ (p65) was then detected by measuring absorbance values at 450 nm with an Infinite® 200 PRO NanoQuant plate reader (Tecan, Mannedorf, Switzerland). Values were then normalized on the respective amount of loaded protein.
4
Cryptosporidium parvum Antigen Detection
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A commercial ELISA kit (Cryptosporidium parvum Antigen Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) was used to detect C. parvum in the diarrheal feces. The diagnostic test was performed according to the manufacturer’s instructions, and in each experiment, positive and negative controls provided by the manufacturer were included.
To determine positivity, optical density (OD) was evaluated at 450 nm using the Infinite® 200 PRO NanoQuant plate reader (Tecan), and the sample-to-positive ratio (S/P) was calculated as manufacturer’s instruction: . The S/P greater than 20 was considered to be a positive result.
To determine positivity, optical density (OD) was evaluated at 450 nm using the Infinite® 200 PRO NanoQuant plate reader (Tecan), and the sample-to-positive ratio (S/P) was calculated as manufacturer’s instruction: . The S/P greater than 20 was considered to be a positive result.
5
Quantifying Oxidative Stress Response
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Reactive oxygen species (ROS) production was quantitated using CellROX® Oxidative Stress Reagents (Thermo Fisher) a fluorogenic probe, according to the manufacturer instructions. 7 × 104 shCTRL and shANK cells were seeded in octuplicate into a 96-well plate, and the plate was incubated for 24 h. Cells were incubated for another 48 h with and without TRA (21 μg/mL), then 100 μL of normal media and CellROX reagent (2.5 mM) were added in each well and plates were incubated for 1 h at 37°C. Green fluorescence values at 520 nm were measured with the Infinite® 200 PRO NanoQuant plate reader (Tecan). Values were then normalized on the respective untreated cells.
6
Assessing Cellular Viability via Resazurin Reduction
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Resazurin reduction capacity of cells present in the explants was assessed using the PrestoBlue Cell Viability Reagent (A13262, Invitrogen). The active ingredient of PrestoBlue reagent (resazurin) is a non-toxic and non-fluorescent dye, that when in contact with a viable cell is reduced, becoming red-fluorescent resorufin76 (link). At days 0, 10, 20 and 30 of each OvC-PDE culture analysed (N = 7–8), 1 mL of culture suspension (on average, 5 explants) was collected in triplicate, and incubated with PrestoBlue reagent (diluted 1:10) during 1 h at 37 °C. After this, supernatants of triplicates were collected to a 96-well black fluorescence reading plate (Corning) and fluorescence reading was performed (Infinite 200 PRO NanoQuant plate reader, TECAN), setting the excitation wavelength at 570 nm and emission wavelength at 590 nm.
7
DNA Extraction and Quantification from Blood
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Genomic DNA was extracted from venous blood samples (3 ml) using a Maxwell DNA Blood Kit (Promega, WI, USA). The extracted genomic DNA was quantified using an Infinite 200 Pro NanoQuant Plate Reader (TECAN, Mannedorf, Switzerland). DNA purity was determined by calculating the ratio of absorbance at 260 and 280 nm.
8
Osteogenic Differentiation Assessment via ALP
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Alkaline Phosphates (ALP) activity was used to measure osteogenic differentiation. ß-TCP scaffold + cells were treated with lysis buffer (400 mM potassium phosphate buffer, 0.01 mM EDTA, 2% triton X-100, pH = 7), and 3 freeze-thaw cycles followed by sonication (45 KHz, 10 minutes, + 4 °C). Cell lysates were collected in fresh micro tubes and processed for ALP activity measurements. ALP assay was performed on all groups at days 3, 7, 14, and 21 according the manufacture’s protocol (SensoLyte pNPP Alkaline Phosphatase Detection kit, Anaspec Inc, CA, USA). Absorbance was measured at 405 nm using Infinite 200PRO NanoQuant plate reader (Tecan, Germany). ALP measurements were normalized against cell number.
9
Osteogenic Alkaline Phosphatase Quantification
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As a marker of osteogenesis, ALP expression was assessed on day 7 of osteogenic culture according to the manufacturer’s protocol (Sensolyte pNPP Alkaline Phosphatase Detection Kit, Anaspec Inc., Köln, Germany). Granules with seeded cells were washed twice with 1x assay buffer and cells were lysed with lysis buffer (0.02% Triton X-100 in 1× assay buffer). Cell lysates were collected into fresh micro tubes and processed for ALP activity measurements. Absorbance was measured at 405 nm by means of Infinite 200PRO NanoQuant plate reader (Tecan, Crailsheim, Germany). The absolute ALP value for each sample was calculated against an alkaline phosphatase standard curve.
10
Epididymal Sperm DNA Methylation Analysis
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Global DNA methylation of epididymal sperm was detected using an ELISA reaction with a monoclonal antibody sensitive and specific for 5-methylcytosine (5-mC) and a horseradish peroxidase conjugate as secondary antibody (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA). The level of 5-mC in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). An aliquot of the sample containing 100 ng DNA was added to the 5-mC coating buffer and brought to a final volume of 100 μL. All samples and controls were denatured at 98 °C for 5 min in a thermocycler and immediately cooled on ice for 10 min. Controls and samples were added to the ELISA plate and incubated at 37 °C for 1 h. After discarding the coating buffer, the wells were washed three times with the 5-mC ELISA buffer and incubated again with 200 μL of 5-mC ELISA buffer at 37 °C for 30 min. The buffer was discarded from the wells and the samples were incubated with the antibody mix at 37 °C for 1 h. The antibody mix consisted of the 5-mC ELISA Buffer, anti-5-methylcytosine and the secondary antibody in a ratio 1:2000:1000. After incubation, the antibody mix was discarded and a 100 μL HRP were added to each well. The absorbance was measured in duplicate at 405 nm using TECAN reader (Infinite® 200 PRO NanoQuant Plate Reader). The results are expressed as % 5-mC per total C.
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