epmotion 5075, by Eppendorf - Product details

1

Optimized DNA Extraction from Swab Samples

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Catch-All Swabs and accompanying tissue were transferred to 0.7 mm garnet bead tubes (MoBio # 13123-50) using sterile forceps, and total DNA was extracted using a modified protocol for the MoBioPowerMag Soil DNA Isolation Kit, optimized for use with the Eppendorf epMotion (MoBio # 27100-4-EP). Prior to bead beating, 750 μL of the RNase A/Bead Solution and 60 μL of the lysis solution were added to tubes, which were then incubated at 70°C for 10–30 minutes. Cells were then lysed using a MP FastPrep-24 at 6.5 m/s for 60 seconds. Tubes were pelleted twice for 10 minutes at room temperature and 21×10³×g to ensure complete removal of inorganic swab material. The standard protocol utilizing an Eppendorf epMotion 5075 was then followed from the inhibitor removal technology step onward. DNA was eluted in MagnaClear Elution Buffer and stored at −20 °C until library preparation.

Sprockett D.D., Ammons C.G, & Tuttle M.S. (2015). Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 23(5), 765-771.

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2

Genome-wide SNP Genotyping of Twin Pairs

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Genomic DNA was extracted from two 4.7 mm card punches of each ANBS using the Beckman GenFind V3 kit and Eppendorf EpMotion 5075 (Eppendorf AG, HH, Germany). DNA concentrations ranged from 1.41–9.86 ng/μL (median 6.62 ng/μL) and ranged in volume from 32 to 50 μL (median 40 μL). Samples were subsequently randomized and submitted to ThermoFischer Scientific for analysis using the Axiom Precision Medicine Diversity Array (PMDA) genome-wide single-nucleotide polymorphism (SNP) array (ThermoFischer, Waltham, MA, USA). Zygosity status was subsequently assessed using an identity-by-descent analysis in PLINK (version 1.90) based on PMDA array, with 43 twin pairs confirmed to be monozygotic with pi-hat values ranging 0.9941–0.9998. The remaining 43 twin pairs were determined to be dizygotic, with pi-hat values ranging 0.4238–0.6116, and removed from further analysis.

Nickels E.M., Li S., Myint S.S., Arroyo K., Feng Q., Siegmund K.D., de Smith A.J, & Wiemels J.L. (2022). DNA methylation at birth in monozygotic twins discordant for pediatric acute lymphoblastic leukemia. Nature Communications, 13, 6077.

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3

Cytotoxicity Evaluation of Nucleoside Analogs

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Stable cell lines were plated 2.025×10^3 cells/well in 96 well plates using either the Beckman 1000 automated work station or the Eppendorf epMotion 5075 (Eppendorf, Hamburg, Germany). Twenty-four hours after plating, cells were treated with varying drug concentrations (10⁻³ M to 10⁻¹⁰ M or 10⁻⁴ M to 10⁻¹¹ M) of individual nucleoside analogs (Cladribine, Gemcitabine, Fluorouracil (5-FU)) for 96 hours at 37°C. Viability was then assessed by exposure to 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyletetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, Missouri, USA) as previously described [17 ]. Data from six wells per drug concentration for each run were averaged and plotted in cytotoxicity curves (Kaleidagraph 4.5, Synergy Software, Reading, Pennsylvania, USA). IC50 values were calculated as the concentration of drug necessary to produce 50% inhibition of cell growth compared to untreated control cells. Median IC50 values for each experiment are provided in the supplementary information (Supplementary Tables 1-3).

Saliba J., Zabriskie R., Ghosh R., Powell B.C., Hicks S., Kimmel M., Meng Q., Ritter D.I., Wheeler D.A., Gibbs R.A., Tsai F.T, & Plon S.E. (2016). Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside analogs. Pharmacogenetics and genomics, 26(6), 271-279.

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4

Quantifying Neutrophil NETosis by Sytox Green Assay

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NETosis was quantified from freshly isolated human neutrophils using a Sytox Green plate reader assay, as described in previous papers.^{69 ,70 (link)} Using a robotic pipetting system, epMotion5075 (Eppendorf North America, Hauppauge, NY, USA), 20,000 cells per well were seeded in a 384-well black, flat, clear-bottom plate (Corning, #3762). The same robotic pipetting system was used to then add 1 µM Sytox Green, a cell impermeable nucleic acid stain, to each well. The plate was split up into 3 groups with 6 wells per group: (i) human neutrophils with 1 nM PMA; (ii) human neutrophils with 1 nM PMA with Heparin (100 IU/mL); (iii) human neutrophils with Heparin (100 IU/mL). The plate was loaded into Cytation 5 plate reader (BioTek-U.S., Winooski, VT, USA), set at 37°C and supplied with 5% CO₂ with a filter setting of 485 nm (excitation) / 527 nm (emission). Kinetic fluorescence intensity was measured every 20 min over 12 hours and analyzed with Gen5 software.

An S., Raju I., Surenkhuu B., Kwon J.E., Gulati S., Karaman M., Pradeep A., Sinha S., Mun C, & Jain S. (2019). Neutrophil Extracellular Traps (NETs) contribute to Pathological Changes of ocular Graft-vs.-Host Disease (oGVHD) Dry Eye: Implications for novel Biomarkers and Therapeutic Strategies. The ocular surface, 17(3), 589-614.

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5

Protein Quantification and Digestion

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Protein concentration was quantified by Micro BCA Assay (ThermoFisher). 13.5 μg of protein of FFPE was transferred to a deep-well plate for processing on an epMotion 5075 liquid handler (Eppendorf, Enfield, CT). Lysates were reduced in ~20 mM TCEP per mg protein for 30 minutes at 37°C with shaking, followed by alkylation with ~40 mM IAM per mg protein in the dark at room temperature. Lysates were then diluted 10x with 200 mM TRIS (pH 8.0), 400 fmol of stable isotope-containing synthetic peptide standards (Vivitide) were spiked into each sample, and Lys-C (Wako) was added to lysates at 1:35 (enzyme:protein) ratio by mass and incubated for 2 hours at 37°C with mixing at 600 RPM (Thermomixer, Eppendorf). After 2 hours, trypsin (Promega) was added at 1:70 enzyme:protein. Digestion was carried out overnight at 37°C with mixing at 600 RPM. After 16 hours, the reaction was quenched with formic acid (FA; final concentration 1% by volume).

Chowdhury S., Kennedy J.J., Ivey R.G., Murillo O.D., Hosseini N., Song X., Petralia F., Calinawan A., Savage S.R., Berry A.B., Reva B., Ozbek U., Krek A., Ma W., da Veiga Leprevost F., Ji J., Yoo S., Lin C., Voytovich U.J., Huang Y., Lee S.H., Bergan L., Lorentzen T.D., Mesri M., Rodriguez H., Hoofnagle A.N., Herbert Z.T., Nesvizhskii A.I., Zhang B., Whiteaker J.R., Fenyo D., McKerrow W., Wang J., Schürer S.C., Stathias V., Chen X.S., Barcellos-Hoff M.H., Starr T.K., Winterhoff B.J., Nelson A.C., Mok S.C., Kaufmann S.H., Drescher C., Cieslik M., Wang P., Birrer M.J, & Paulovich A.G. (2023). Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer. Cell, 186(16), 3476-3498.e35.

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6

Protein Quantification and Enzymatic Digestion

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Protein concentration was quantified by Micro BCA Assay (ThermoFisher). 100 μg of protein of FFPE or 500 μg frozen tissue lysates (diluted to 2 mg/mL in lysis buffer) was transferred to a deep-well plate for processing on an epMotion 5075 liquid handler (Eppendorf, Enfield, CT). Lysates were reduced in 16.5 mM TCEP per mg protein for 30 minutes at 37°C with shaking, followed by alkylation with 36 mM IAM per mg protein in the dark at room temperature. Lysates were then diluted with 200 mM TRIS (pH 8.0), to a urea concentration of < 1M before Lys-C (Wako) was added to lysates at 1:50 (enzyme:protein) ratio by mass and incubated for 2 hours at 37°C with mixing at 600 RPM (Thermomixer, Eppendorf). After 2 hours, trypsin (Promega) was added at 1:100 enzyme:protein. Digestion was carried out overnight at 37°C with mixing at 600 RPM. After 16 hours, the reaction was quenched with formic acid (FA; final concentration 1% by volume).

Petit M.M., Fradelizi J., Golsteyn R.M., Ayoubi T.A., Menichi B., Louvard D., Van de Ven W.J, & Friederich E. (2000). LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity. Molecular Biology of the Cell, 11(1), 117-129.

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7

Protein Quantification and Enzymatic Digestion

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Protein concentration was quantified by Micro BCA Assay (ThermoFisher). 100 μg of protein of FFPE or 500 μg frozen tissue lysates (diluted to 2 mg/mL in lysis buffer) was transferred to a deep-well plate for processing on an epMotion 5075 liquid handler (Eppendorf, Enfield, CT). Lysates were reduced in 16.5 mM TCEP per mg protein for 30 minutes at 37°C with shaking, followed by alkylation with 36 mM IAM per mg protein in the dark at room temperature. Lysates were then diluted with 200 mM TRIS (pH 8.0), to a urea concentration of < 1M before Lys-C (Wako) was added to lysates at 1:50 (enzyme:protein) ratio by mass and incubated for 2 hours at 37°C with mixing at 600 RPM (Thermomixer, Eppendorf). After 2 hours, trypsin (Promega) was added at 1:100 enzyme:protein. Digestion was carried out overnight at 37°C with mixing at 600 RPM. After 16 hours, the reaction was quenched with formic acid (FA; final concentration 1% by volume).

Chowdhury S., Kennedy J.J., Ivey R.G., Murillo O.D., Hosseini N., Song X., Petralia F., Calinawan A., Savage S.R., Berry A.B., Reva B., Ozbek U., Krek A., Ma W., da Veiga Leprevost F., Ji J., Yoo S., Lin C., Voytovich U.J., Huang Y., Lee S.H., Bergan L., Lorentzen T.D., Mesri M., Rodriguez H., Hoofnagle A.N., Herbert Z.T., Nesvizhskii A.I., Zhang B., Whiteaker J.R., Fenyo D., McKerrow W., Wang J., Schürer S.C., Stathias V., Chen X.S., Barcellos-Hoff M.H., Starr T.K., Winterhoff B.J., Nelson A.C., Mok S.C., Kaufmann S.H., Drescher C., Cieslik M., Wang P., Birrer M.J, & Paulovich A.G. (2023). Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer. Cell, 186(16), 3476-3498.e35.

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8

Protein Quantification and Digestion

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Protein concentration was quantified by Micro BCA Assay (ThermoFisher). 13.5 μg of protein of FFPE was transferred to a deep-well plate for processing on an epMotion 5075 liquid handler (Eppendorf, Enfield, CT). Lysates were reduced in ~20 mM TCEP per mg protein for 30 minutes at 37°C with shaking, followed by alkylation with ~40 mM IAM per mg protein in the dark at room temperature. Lysates were then diluted 10x with 200 mM TRIS (pH 8.0), 400 fmol of stable isotope-containing synthetic peptide standards (Vivitide) were spiked into each sample, and Lys-C (Wako) was added to lysates at 1:35 (enzyme:protein) ratio by mass and incubated for 2 hours at 37°C with mixing at 600 RPM (Thermomixer, Eppendorf). After 2 hours, trypsin (Promega) was added at 1:70 enzyme:protein. Digestion was carried out overnight at 37°C with mixing at 600 RPM. After 16 hours, the reaction was quenched with formic acid (FA; final concentration 1% by volume).

Petit M.M., Fradelizi J., Golsteyn R.M., Ayoubi T.A., Menichi B., Louvard D., Van de Ven W.J, & Friederich E. (2000). LPP, an Actin Cytoskeleton Protein Related to Zyxin, Harbors a Nuclear Export Signal and Transcriptional Activation Capacity. Molecular Biology of the Cell, 11(1), 117-129.

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9

SAV3 Detection via Q_nsp1 Assay

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The SAV3 strain was detected using the Q_nsp1 assay [25 (link)]. This broad spectrum assay detects all known SAV subtypes using primers and probe (Table 1) with final concentrations of 500 and 300 nM, respectively, and amplifies a conserved region in the 5’ end of the nsp1 gene, giving amplicons of 107 bp. Extracted RNA was automatically pipetted by Eppendorf epMotion^® 5075 (Eppendorf, Hamburg, Germany) in duplicates, analyzed by RT-qPCR on an AriaMx machine (Agilent Technologies, Santa Clara, CA, USA), and evaluated with the Agilent AriaMx Real-Time PCR software (version 1.7). Each plate included a negative control sample and an inter-plate calibrator of pure SAV3 RNA, which were both run in duplicates. The cut-off quantification cycle (Cq) value was set to 40; samples with values below this Cq in duplicates were considered positive. Samples with only one positive parallel were rerun and considered positive only with positive duplicates. The template volume was 2.0 μL RNA in a total reaction volume of 20 μL, and the RT-qPCR kit used was TaqMan^® Fast Virus 1-Step Master Mix (Applied Biosystems®, Foster City, CA, USA). The thermal program comprised reverse transcription for 5 min at 50 °C and enzyme activation for 2 min at 95 °C, followed by 45 cycles of 15 s at 94 °C and 40 s at 60 °C.

Tartor H., Bernhardt L.V., Mohammad S.N., Kuiper R, & Weli S.C. (2023). In Situ Detection of Salmonid Alphavirus 3 (SAV3) in Tissues of Atlantic Salmon in a Cohabitation Challenge Model with a Special Focus on the Immune Response to the Virus in the Pseudobranch. Viruses, 15(12), 2450.

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10

Real-Time PCR Analysis of Gene Expression

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Real-Time PCR was performed using primers specific for Tgfb3, Tgfbr2, Il4, tPA, Mapk14, Icam-1 and Vcam-1 from SA Biosciences (Qiagen, Valencia, CA). RT-PCR reactions were performed according to manufacturer’s instructions using RT2 SYBR Green (SA Biosciences). All assays including no template controls were done in triplicate. One stable housekeeping gene was used as internal control (B2m). Samples were placed onto 96-well plates using an epMotion® 5075 automated pipetting system (Eppendorf, Hauppauge, NY) and run on a ViiA 7 Real-Time PCR System (Life Technologies, Grand Island, NY) using manufacturer recommended cycling conditions. Data were analyzed using comparative Ct method (ΔΔCt method).

Bianchi E., Boekelheide K., Sigman M., Lamb D.J., Hall S.J, & Hwang K. (2016). Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-β Signaling Pathway. PLoS ONE, 11(4), e0153968.

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Epmotion 5075

Lab products found in correlation

71 protocols using epmotion 5075

Optimized DNA Extraction from Swab Samples

Genome-wide SNP Genotyping of Twin Pairs

Cytotoxicity Evaluation of Nucleoside Analogs

Quantifying Neutrophil NETosis by Sytox Green Assay

Protein Quantification and Digestion

Protein Quantification and Enzymatic Digestion

Protein Quantification and Enzymatic Digestion

Protein Quantification and Digestion

SAV3 Detection via Q_nsp1 Assay

Real-Time PCR Analysis of Gene Expression

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