Drop plate

Manufactured by Thermo Fisher Scientific

Sourced in United States, Finland

The µDrop plate is a multi-well plate designed for high-throughput spectrophotometric measurements of small sample volumes. It features a specialized well geometry and optical path that enables accurate absorbance readings from microliter-scale samples.

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Lab products found in correlation

16 protocols using drop plate

Quantifying CDV Loads and Cytokines

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To quantify CDV loads and cytokine responses at the molecular level, RT-qPCR analysis (38 slices derived from 4 dogs) was performed. RNA was isolated and purified from frozen PCLSs using the RNeasy^® Mini Kit (Qiagen, Hilden, Germany) as described [38 (link)]. Measurement of the optical density at 260 nm with a spectrophotometer (Multiskan™ GO microplate spectrophotometer, µDrop™ plate, SkanIt™ software version 5.0.0.42, Thermo Fisher Scientific, Braunschweig, Germany) allowed calculation of the obtained RNA amount and absorbance at 260 nm and 280 nm was used to assess the purity of RNA.
Total RNA was transcribed into complementary DNA (cDNA) using the Omniscript^® Reverse Transcription Kit (Qiagen), RNaseOUT™ Recombinant Ribonuclease Inhibitor (Invitrogen, Carlsbad, CA, USA), and random primers (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions as described [38 (link)].
Primer sequences and plasmids for generation of standard dilutions and qPCR products (Eurofins Genomics, Ebersberg, Germany) were taken from the literature and have been described in detail in a previous publication [38 (link)]. Standard dilutions were either generated from extracted PCR products or plasmids, and RT-qPCR analysis using the AriaMx Real-Time PCR System (Agilent Technologies; Agilent Aria software version 1.71) was performed as described [38 (link)].

Chludzinski E., Ciurkiewicz M., Stoff M., Klemens J., Krüger J., Shin D.L., Herrler G, & Beineke A. (2023). Canine Distemper Virus Alters Defense Responses in an Ex Vivo Model of Pulmonary Infection. Viruses, 15(4), 834.

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Quantitative RT-PCR Gene Expression Analysis

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NucleoSpin RNA II kit (MACHEREY-NAGEL, Düren, Germany) and the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) were used to prepare RNA and synthesize cDNA. RNA concentration and quality were measured with Varioskan LUX Multimode Microplate Reader and µDrop Plate (Thermo Fisher Scientific). Quantitative RT-PCR was performed with 1 ng cDNA and SYBR Green primer pairs (Sigma-Aldrich) in 10 μL reaction on the QuantStudio Real-Time PCR machine (Thermo Fisher Scientific). Relative gene expression was calculated with the 2^−ΔCt method (26 (link)). Primer pairs are listed in Table 1.

Kulyté A., Aman A., Strawbridge R.J., Arner P, & Dahlman I.A. (2022). Genome-Wide Association Study Identifies Genetic Loci Associated With Fat Cell Number and Overlap With Genetic Risk Loci for Type 2 Diabetes. Diabetes, 71(6), 1350-1362.

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Cerebrum and Spleen RNA Isolation

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For RNA isolation, snap frozen tissue of the cerebrum was homogenized in 1 ml QIAzol lysis reagent (Qiagen, Hilden, Germany) with Omni Tip PCR Tissue Homogenizing Kit (Süd-Laborbedarf GmbH, Gauting, Germany). Subsequently, homogenates were treated with RNeasy Lipid tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Likewise, RNA isolation of snap frozen splenic tissue has been performed using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The purity and amount of RNA was measured with a Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) using a µDrop plate (Thermo Fisher Scientific, Waltham, MA, USA) and SkanIt software (version 3.2.1.4 RE, Thermo Fisher Scientific, Waltham, MA, USA)⁹⁴. Equal amounts of RNA were translated into cDNA using Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany), RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and random primers (Promega Corporation, Madison, WI, USA).

Stoff M., Ebbecke T., Ciurkiewicz M., Pavasutthipaisit S., Mayer-Lambertz S., Störk T., Pavelko K.D., Baumgärtner W., Jung K., Lepenies B, & Beineke A. (2021). C-type lectin receptor DCIR contributes to hippocampal injury in acute neurotropic virus infection. Scientific Reports, 11, 23819.

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Microarray-based Gene Expression Analysis

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Total RNA was isolated from cell culture using NucleoSpin RNA (Macherey-Nagel, Hoerdt, France), according to the manufacturer’s instructions. RNA quantity and quality were measured on a MultiSkan GO spectrophotometer with a µDrop Plate (ThermoFisher Scientific, France). RNA integrity was assessed on a 2100 Bioanalyzer (Agilent Technologies France, Les Ulis, France) using RNA 6000 Nano (Agilent Technologies), according to the manufacturer’s instructions. RNA integrity numbers above 8 were considered suitable for microarray analysis. Two-color microarray-based gene expression analysis was performed, according to the manufacturer’s instructions (Agilent Technologies). Briefly, 100 ng of total RNA were amplified and labeled using a Low input Quick Amp Labeling kit, Two-color (Agilent Technologies), and hybridized to a Human Gene Expression 4 × 44 K v2 Microarray (design ID 026652, Agilent Technologies). Slides were scanned on a G2505C Microarray Scanner (Agilent Technologies). Raw data were extracted and Lowess normalized using Feature Extraction software (v. 10.7.3, Agilent Technologies), and analyzed using GeneSpring GX software (v. 13.1.1, Agilent Technologies). Microarray probes with a signal that is not positive and significant or not above the background were filtered out.

Aury-Landas J., Bazille C., Allas L., Bouhout S., Chesneau C., Leclercq S., Boumédiene K, & Baugé C. (2017). Anti-inflammatory and chondroprotective effects of the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A, in human articular chondrocytes. Scientific Reports, 7, 6483.

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Isolation and Analysis of RNA from Whole Blood

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Prior to morning feeding, blood samples (n = 56) were collected
on the first day of the trial (before supplementation began) and on d 3, 5, 10,
14, 21 and 28 of supplementation. Approximately 3 ml of whole blood was
collected via jugular venipuncture into Tempus Blood RNA Tubes (Cat no 4342792,
Life Technologies, Carlsbad, CA, USA). Immediately after collection, tubes were
shaken vigorously for 15 s and stored at –20°C until RNA isolation (less than
2 mo). RNA was isolated using the Tempus Spin RNA Isolation kit (Cat no 4380204,
Life Technologies); upon completion, RNA samples were stored at –80°C until
reverse transcription.
RNA was evaluated for purity and concentration using a Thermo Scientific
Multiskan Go microplate spectrophotometer (Cat no. 51119300; Thermo Fisher
Scientific, Carlsbad, CA, USA) and a µDrop Plate (cat no N12391, Thermo Fisher
Scientific). Only RNA with an absorbance ratio (260 nm: 280 nm) above 2.0 was
used (n = 54). One μg total RNA was completely used as a
template for cDNA synthesis in a RT² First Strand Kit following the
manufacturer’s protocol (cat no 330401; QIAGEN, Valencia, CA, USA). Upon
completion, cDNA was stored at –20°C until use.

Armstrong S.A., McLean D.J., Bionaz M, & Bobe G. (2019). A natural bioactive feed additive alters expression of genes involved in inflammation in whole blood of healthy Angus heifers. Innate Immunity, 26(4), 285-293.

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Isolation and cDNA Synthesis from RAW 264.7 Cells

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Trizol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from the RAW 264.7 macrophages. The extraction procedure followed manufactures instructions. RNA purity was measured (µDrop plate, Thermo Scientific, Waltham, MA, USA) and produced cDNA via reverse-transcription with a first-strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan Japan) following given instructions. (Primers are indicated in Table 4)

Jayawardena T.U., Sanjeewa K.K., Nagahawatta D.P., Lee H.G., Lu Y.A., Vaas A.P., Abeytunga D.T., Nanayakkara C.M., Lee D.S, & Jeon Y.J. (2020). Anti-Inflammatory Effects of Sulfated Polysaccharide from Sargassum swartzii in Macrophages via Blocking TLR/NF-Κb Signal Transduction. Marine Drugs, 18(12), 601.

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Exosome Isolation from Conditioned Medium

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Total Exosome Isolation Reagent (Thermo Fisher Scientific) was used to isolate exosomes released from ASCs cultured in CM. After 72 h of culture, the conditioned medium was replaced with a growth medium supplemented with 10% exosome-depleted FBS (Thermo Fisher Scientific). The medium was harvested after a subsequent 24 h culture, centrifuged, and mixed with Total Exosome Isolation Reagent. After overnight incubation, the samples were centrifuged at 1 × 10⁴× g for 1 h. Pelleted exosomes were resuspended in PBS. Protein concentration was measured with the µDrop Plate using a Mutliskan Sky microplate reader (Thermo Fisher Scientific).

Maj M., Kaźmierski Ł., Balik K., Kowalska K., Gackowska L., Bajek A, & Drewa T. (2022). Bladder Cancer Cells Exert Pleiotropic Effects on Human Adipose-Derived Stem Cells. Life, 12(4), 549.

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Whole Genomic DNA Extraction Protocol

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Whole genomic DNA (gDNA) was extracted by lysing a maximum of 5 × 10⁶ cells for 18–24 h at 55 °C in 487.5 µL TENS buffer (10 mM Tris–HCl (Sigma cat. T3253) pH 8.0, 25 mM EDTA (Sigma cat. 324506) pH 7.5, 100 mM NaCl (Sigma cat. S1679), 0.5% SDS (Sigma cat. 71736)) with 12.5 µL of proteinase K (20 mg/mL; Sigma cat. 70663-4). Proteins were precipitated using 250 µL of 6 M NaCl followed by centrifugation for 5 min at 12,000 g. The supernatant was recovered, and gDNA was precipitated with 900 µL isopropanol followed by centrifugation at 12,000 g for 10 min. The pellet was collected and washed with cold 70% EtOH and allowed to dry. gDNA was resuspended in 50 µL of TE buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA pH 8.0) and purity was assessed using the Multiskan SkyHigh Microplate Spectrophotometer and µdrop plate (Thermo Fisher Scientific). Sample purity was measured by determining the 260/280 nm and 260/230 nm absorption ratios with samples achieving 1.7–2.0 and 2.0–2.2, respectively, being used.

Cuesta-Gomez N., Verhoeff K., Dadheech N., Dang T., Jasra I.T., de Leon M.B., Pawlick R., Marfil-Garza B., Anwar P., Razavy H., Zapata-Morin P.A., Jickling G., Thiesen A., O’Gorman D., Kallos M.S, & Shapiro A.M. (2023). Suspension culture improves iPSC expansion and pluripotency phenotype. Stem Cell Research & Therapy, 14, 154.

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Protein Extraction and Tryptic Digestion from Biofilm and Planktonic Cells

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The cells were suspended in 100 mM Tris-HCl (pH 6.8); from biofilm cultures cell-samples were obtained by scraping and from the planktonic cultures by centrifugation (3 min, 8000× g, +4 °C). Washed cells collected by centrifugation (3 min, 8000× g, +4 °C) were suspended gently in 90 μL of TEAB (triethylammonium bicarbonate) containing 10% sucrose [15 (link)]. Tryptic digestions were initiated with 55 ng/µL of sequencing grade-modified porcine trypsin (Promega, Madison, WI, USA) and digestions were incubated at 37 °C for 20 min. Released peptides and trypsin were recovered by filtration through a 0.2 µm acetate membrane (Costar^® Spin-X Centrifuge Tube Filter, Corning Inc., Corning, NY, USA) by centrifugation (7000× g, 3 min, +4 °C). Flow-troughs containing the released peptides and trypsin were further incubated for 16 h at 37 °C. Digestions were stopped by adding trifluoroacetate (TFA) to a final concentration of 0.6% (v/v). The peptide concentrations were measured with Multiskan Sky using the µDrop plate (Thermo Fisher Scientific; Vantaa, Finland).

Savijoki K., Miettinen I., Nyman T.A., Kortesoja M., Hanski L., Varmanen P, & Fallarero A. (2020). Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus. Microorganisms, 8(1), 106.

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Quantification of Gene Expression from Tumor Tissues

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Total RNA was extracted from frozen tumor tissue samples using TRIzol^® reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). RNA amounts and purity were determined on a microplate reader (Varioskan Lux, ThermoScientific, Waltham, MA, USA) with a µDrop^® plate (ThermoScientific, Waltham, MA, USA) and RNA integrity was controlled on 1.2% agarose gel using the FlashGel electrophoresis system (Lonza, Rockland, ME, USA). Reverse transcription reaction was carried out on 1 μg of total RNA using the iScript Supermix (Bio-Rad, Hercules, CA, USA). QPCR was performed on a CFX-96 Real Time System (Bio-Rad, Hercules, CA, USA) using Sybrgreen method (SsoAdvanced Universal Sybr^® Green Supermix, Bio-Rad, Hercules, CA, USA). The expression of target genes was normalized to reference genes (HPRT1, RPL4 and RPL19) and the relative expression was calculated using the ΔΔCt method (Table 2).

Gouez M., Rébillard A., Thomas A., Beaumel S., Matera E.L., Gouraud E., Orfila L., Martin B., Pérol O., Chaveroux C., Chirico E.N., Dumontet C., Fervers B, & Pialoux V. (2024). Combined effects of exercise and immuno-chemotherapy treatments on tumor growth in MC38 colorectal cancer-bearing mice. Frontiers in Immunology, 15, 1368550.

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