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Latest AI and User-Annotated Protocols for Enhanced Reproducibility

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Last 30 annotated protocols

1
Cassava Flour Moisture Content Determination
Not available on PMC !
A PCE Instruments' COLOR METER model PCE-CSM 2 (Color Tec Associates, Inc., 28 Center Street, Clinton, NJ 08809) was used to determine the color variation of the dried samples using the three different drying methods described by Alamu et al. (2020) . The L * , a * , and b * readings were recorded in an Excel sheet. The color meter operates on the Commission Internationale de l'Eclairage (CIE) L * , a * , b * color scheme. The maximum for L * is 100, representing a perfect reflecting diffuser, the value for the standard white paper. The positive a * value is red, the negative a * value is green, the positive b * value is yellow, and the negative b * indicates blueness. The total color difference, E * , was calculated using the L * , a * , and b * values. The E * is a single value considering the differences between the sample's and standard's L * , a * , and b * .
. . Fundamental chemical analysis: proximate (moisture, ash, fat, amylose, total starch, and sugar) composition determinations . . . Moisture content
The cassava flour's moisture content (MC) was determined by weighing 5 g of the cassava samples in a cleaned and pre-weighed moisture can and placed in an oven at 103 • C for 16 h until the constant weight was obtained (AOAC, 2006) . The % MC was estimated using the equation shown below:
Where M 2 = weight in (g) of dish, lid, and sample after drying M 1 = weight in (g) of dish, lid, and sample before drying M 0 = weight in (g) of dish and lid only.
Alamu E.O., Manda N., Ntawuruhunga P., Abass A., & Maziya‐Dixon B. (2023). Elite cassava clones (Manihot esculenta) grown in Zambia: effects of drying techniques on their chemical, functional, and pasting properties. Frontiers in Sustainable Food Systems, 7.
Publication 2023
2
RNA Extraction and qRT-PCR Analysis
Not available on PMC !
Harvested cells were washed with PBS and lysed with Trizol (TaKaRa, Japan). Detailed procedures were followed form the instruction of TaKaRa. RNA was reverse-transcribed by PrimeScriptTM RT Master Mix Kit (TaKaRa, Japan). TB Green Premix Ex Taq II Kit (TakaRa, Japan) was used for quantitative real-time PCR and analyzed by Light Cycler 480 (Roche, Germany).
Zhong G., Wang Y., Zhou H., Fu Z., Chen Z., & Yao T. (2022). TGF-β signaling and N6-methyladenosine with ciRs-7 in cervical cancer metastasis.
Publication 2022
3
Isolation and Identification of Lactobacillus Strains from Algerian Poultry
The lactobacilli strains were isolated from gizzard contents of Algerian local poultry. Decimal dilution of these samples were mixed with MRS medium and incubated at 37°C for 48 h under anaerobiosis (7 ). Selected colonies were picked from the higher dilutions and sub-cultured in MRS broth. The identification of the isolates was performed according to the criteria of Bergey’s Manual of Determinative Bacteriology and using the methods and criteria of Sharpe (8 ). The isolates were initially subjected to Gram staining and catalase test (3% H2O2). Only the Gram positive, catalase negative isolates were further identified. Growth at different temperatures was determined in MRS broths (10°C, 15°C, 40°C and 45°C). Hydrolysis of arginine was also recorded. The fermentative type was determined on agar.
The ability of the isolated strains to produce acid from different carbohydrates was determined by API 50 CHL test kits (BioMerieux, S.A., France). The results were loaded onto the API system software, which used the phenotypic data to predict a species identity (%) for each isolate.
, & Idoui T. (2014). Probiotic properties of Lactobacillus strains isolated from gizzard of local poultry. Iranian Journal of Microbiology, 6(2), 120-126.
Publication 2014
Acids Agar Anaerobiosis Arginine Carbohydrates Catalase Fermentation Fowls, Domestic Gizzard Hydrolysis Lactobacillus Peroxide, Hydrogen Phenotype Strains Technique, Dilution
4
Quantifying Sourdough Microbial Diversity
Cited 1 time
Five grams of sourdough were supplemented with sterile 0.85% NaCl solution up to a volume of 50 ml. The mixture was then homogenized by vortexing. Decimal dilutions were plated on to both sourdough bacteria (SDB) agar (maltose, 2.0%; yeast extract, 1%; Tween 80, 0.03%, trypticase 0.6%; pH 5.6) and de Man, Rogosa and Sharpe (MRS) agar (Lab M Ltd, UK) with 100 µg/ml cycloheximide (Sigma-Aldrich, USA).
The plates were incubated at the same temperature the sourdough was fermented at (20 or 30°C). Incubation was carried out for 48 h under anaerobic conditions (AnaeroGen, Oxoid). Colony forming units (CFU) were counted from the agar media using suitable dilutions.
For each of the six sourdough samples collected on day 56, 20 colonies were picked from the MRS and SDB agar plates (ten from each medium) for further analysis by rep-PCR. Colony picking was performed in succession from one sector of the plate. On day 56 samples were also plated on Yeast Extract Peptone Dextrose (YPD) agar (dextrose, 2.0%; peptone 2%; yeast extract, 1%) with 100 µg/ml chloramphenicol (Sigma-Aldrich, USA) and incubated at the same temperature the sample was fermented at (20 or 30°C). Ten colonies per sample were picked in succession from YPD agar plates and analyzed using RAPD-PCR.
Bessmeltseva M., Viiard E., Simm J., Paalme T, & Sarand I. (2014). Evolution of Bacterial Consortia in Spontaneously Started Rye Sourdoughs during Two Months of Daily Propagation. PLoS ONE, 9(4), e95449.
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Publication 2014
Agar Bacteria Chloramphenicol Cycloheximide Glucose Maltose Peptones Random Amplified Polymorphic DNA Technique Sodium Chloride Sterility, Reproductive Technique, Dilution trypticase Tween 80 Yeasts
5
Identification of Lactic Acid Bacteria and Yeasts
Not available on PMC !
The identification of lactic acid bacteria was done by phenotypic analysis using the kit API50CHL (BioMérieux, Marcy-l'Etoile, France) according Saeed et al.(2009) and Lu et al. (2008) .The API 50CHL system allows the species identification of LAB according to the biochemical profile of carbohydrate fermentation. Readings were taken after 24 and 48 hours of incubation at 30°C. The identification of yeasts was performed using the kit API 20CAux (BioMérieux, Marcy-l'Etoile, France) according Saeed et al.(2009) and the readings were made after 48 and72 hours of incubation at 30 °C.
Aplevicz K.S., Mazo J.Z., Ilha E.C., Dinon A.Z., & ́Anna E.S.S. (2014). Isolation and characterization of lactic acid bacteria and yeasts from the Brazilian grape sourdough. Brazilian Journal of Pharmaceutical Sciences, 50(2), 321-327.
Publication 2014
6
Lactic Acid Bacteria and Yeast Isolation from Greek Wheat Sourdoughs
A total of 207 lactic acid bacteria and 195 yeast isolates were obtained from thirteen Greek spontaneously fermented wheat sourdoughs [25 (link)]. The lactic acid bacteria isolates were identified as follows: Lactiplantibacillus plantarum (70 isolates); Levilactobacillus brevis (71 isolates); Companilactobacillus paralimentarius (30 isolates); Lvb. zymae (1 isolate); Latilactobacillus curvatus (6 isolates); Ltb. sakei (12 isolates); Leuconostoc citreum (1 isolate); Ln. mesenteroides (1 isolate); Lactococcus lactis (3 isolates); and Fructilactobacillus sanfranciscensis (12 isolates). The yeast isolates were identified as follows: Saccharomyces cerevisiae (161 isolates); Kazachstania humilis (2 isolates); Pichia fermentans (8 isolates); Pi. membranifaciens (18 isolates); and Wickerhamomyces anomalus (6 isolates). All isolates were stored at −20 °C in a Nutrient broth supplemented with 50% glycerol (Applichem, Darmstadt, Germany). Before experimental use, lactic acid bacteria and yeast isolates were grown twice in de Mann, Rogosa, and Sharpe (MRS) broth, and in Brain Heart Infusion (BHI) broth, and their purity was examined through streaking in MRS agar and BHI agar, respectively. All substrates were from LAB M (Lancashire, UK).
Syrokou M.K., Tziompra S., Psychogiou E.E., Mpisti S.D., Paramithiotis S., Bosnea L., Mataragas M., Skandamis P.N, & Drosinos E.H. (2021). Technological and Safety Attributes of Lactic Acid Bacteria and Yeasts Isolated from Spontaneously Fermented Greek Wheat Sourdoughs. Microorganisms, 9(4), 671.
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Publication 2021
Agar Brain Glycerin Heart Kazachstania humilis Lactobacillales Lactobacillus curvatus Lactobacillus paralimentarius Lactobacillus sanfranciscensis Lactococcus lactis Leuconostoc citreum Nutrients Pichia anomala Pichia fermentans Saccharomyces cerevisiae Triticum aestivum Yeast, Dried
7
Characterization of Sourdough Microbiome
The eight mature spontaneous sourdoughs were subjected to analyses in triplicate using M17 (30 °C, 48 h), mostly for coccus-shaped lactic acid bacteria, and SDB, mMRS and MRS5 (30 °C, 48 h), mostly for dominant lactobacilli and Weissella. Slanetz & Bartley agar (spread plate method, 37 °C, 24–48 h) was used for enterococci, Baird-Parker agar (spread plate method, 30 °C, 48 h) for staphylococci and micrococci, VRBGA for total coliforms, and WA and SDA (30 °C, 48 h) for yeasts. For all media, colonies of bacteria and yeasts with different morphologies were picked and isolated from the penultimate dilution, with the exception of the subdominant culturable lactobacilli, which was isolated from 140 mm diameter plates and considering several decimal dilutions. Isolated colonies were cultivated in broth media and then restreaked onto agar medium (MRS for all lactic acid bacteria and SDA for yeasts) until they were purified. Bacterial and yeast isolates were identified by partial sequencing of the 16S rRNA and 26S rRNA genes, respectively [8 (link), 9 (link)]. The identified strains comprising the sourdough biobank were further used upon selection to reconstruct the de novo synthetic microbial communities.
Calabrese F.M., Ameur H., Nikoloudaki O., Celano G., Vacca M., Junior W.J., Manzari C., Vertè F., Di Cagno R., Pesole G., De Angelis M, & Gobbetti M. (2022). Metabolic framework of spontaneous and synthetic sourdough metacommunities to reveal microbial players responsible for resilience and performance. Microbiome, 10, 148.
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Publication 2022
Agar Bacteria Enterococcus Genes Lactobacillales Lactobacillus Microbial Community Micrococcus Ribosomal RNA Genes RNA, Ribosomal, 16S RNA, ribosomal, 26S Staphylococcus Strains Technique, Dilution Weissella Yeast, Dried Yeasts
8
Protein Extraction from Brain Tissue
Cited 2 times
The harvested brain tissues were homogenized on ice using an immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40) plus protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A). The lysates were collected, centrifuged at 12,000 rpm for 10 min, and quantified for total proteins using a bicinchoninic acid protein assay kit (Pierce, Iselin, NJ).
Zhang Y., Zhen Y., Dong Y., Xu Z., Yue Y., Golde T.E., Tanzi R.E., Moir R.D, & Xie Z. (2011). Anesthetic Propofol Attenuates the Isoflurane-Induced Caspase-3 Activation and Aβ Oligomerization. PLoS ONE, 6(11), e27019.
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Publication 2011
Aprotinin bicinchoninic acid Biological Assay Brain Buffers Edetic Acid Immunoprecipitation leupeptin Nonidet P-40 pepstatin Protease Inhibitors Proteins Sodium Chloride Tissues Tromethamine
9
Tumor Spheroid-Immune Cell Co-Culture
2.5x104 TNBC cells were suspended in 100% Matrigel (Corning) in a 48-well plate, and 300μl of the corresponding media were added over the spheroid plugs in each well. For co-culture assays with immune cells, the human spheroids were resuspended in 65-70% of Matrigel and co-cultured with human peripheral blood derived DCs, and 4T1 spheroids were resuspended in 65% of Matrigel and co-cultured with purified bone marrow dendritic cells (BMDCs). Human DCs or mouse BMDCs were labeled with CellTracker Deep Red Dye (ThermoFisher) at a final concentration of 1X in 1ml of complete media. The labeled cells were incubated for 30 minutes at 37˚C. Cells were washed (15,000 RPM 10 minutes at room temperature). The labelled cells were resuspended in complete media, counted, and co-cultured with treated spheroids (1 spheroid:10 immune cells).
Niavarani S.R., St-Cyr G., Daniel L., Lawson C., Giguère H., Alkayyal A.A, & Tai L.H. (2023). Heterologous prime-boost cellular vaccination induces potent antitumor immunity against triple negative breast cancer. Frontiers in Immunology, 14, 1098344.
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Publication 2023
Biological Assay Bone Marrow Cells Cell Culture Techniques Cells Dendrites Homo sapiens matrigel Mus
10
Determining IC50 Using GraphPad Prism
Not available on PMC !
For the determination of the IC50, GraphPad Prism 9 (GraphPad Software, LLC) was used. After 20 hours, the OD600nm absorbance kinetic data were obtained and normalized using the positive control absorbance value as 100% viability and the absorbance value of 1 mM HDC1 as 0% viability. The analysis was then done on the normalized data by nonlinear regression curve fitting. The IC50 value is shown with a 95% confidence interval (CI).
Breine A., Gysel M.V., Elsocht M., Whiteway C., Philippe C., Quinet T., Valček A., Wouters J., Ballet S., & Henst C.V.d. (2021). Antimicrobial activity of a repurposed harmine-derived compound on extensively drug-resistantAcinetobacter baumanniiclinical isolates.
Publication 2021
11
Whole Genomic DNA Extraction Protocol
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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Publication 2023
Buffers Cells Centrifugation Cold Temperature Edetic Acid Endopeptidase K Ethanol Genome Isopropyl Alcohol Proteins Sodium Chloride Tetranitrate, Pentaerythritol Tromethamine
12
Plasma RNA Isolation and Profiling
Ten milliliters of blood sample were drawn from each participant into an EDTA vacutainer tube. The samples were kept in a portable Styrofoam box with ice packs (0–4°C), processed within 6 hours, and stored at −80°C until RNA isolation was conducted.
Total RNA, including miRNA, was isolated and purified from plasma samples using Qiagen’s miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. To minimize cellular contamination, the plasma samples were centrifuged at 16,000×g at 4°C for 10 min and 200 μl supernatant was used for total RNA extraction. To control for variations in the starting material as well as the efficiency of the downstream total RNA extraction, 5 μl each of three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added at concentrations of 1 pg/μl for cel-miR248, 2 pg/μl for osa-miR414, and 4 pg/μl for ath-miR159a. Five microliters of total RNA from 25 μl total yield was used for miRNA expression analysis, which represented the RNA from 40 μl of plasma.
Wu J., Cai H., Xiang Y.B., Matthews C.E., Ye F., Zheng W., Cai Q, & Shu X.O. (2018). Intra-individual Variation of miRNA Expression Levels in Human Plasma Samples. Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals, 23(4), 339-346.
Publication 2018
2',5'-oligoadenylate BLOOD Cells Edetic Acid isolation MicroRNAs Plasma Serum styrofoam
13
Serum miRNA Profiling Protocol
Peripheral blood from each participant was collected and processed for serum extraction. Samples were centrifuged at 3000 g for 10 min at room temperature. Serum was transferred to new EP tubes and stored at − 80 °C for future analysis.
For microarray analysis, total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. miRNA labeling and hybridization were conducted with the miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Santa Clara, CA, USA). For samples in the validation set, miRNAs were isolated from serum using the MiRNeasy Serum/Plasma kit (Qiagen, Valencia, CA, USA). RNA sample quality tests including RNA purity, total amount and integrity tests were performed. RNA purity was checked by the ratio of absorbance at 260 nm and 280 nm, a ratio of above 1.9 was accepted. The total RNA amount of each sample was at least 1 μg. RNA integrity was checked by agarose gel electrophoresis.
Zhang X., Tan J., Chen Y., Ma S., Bai W., Peng Y, & Shi G. (2022). Identification of serum MiRNAs as candidate biomarkers for non-small cell lung cancer diagnosis. BMC Pulmonary Medicine, 22, 479.
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Publication 2022
BLOOD Crossbreeding Electrophoresis, Agar Gel Microarray Analysis MicroRNAs Plasma Serum trizol
14
Quantification of miRNA Expression
Total RNAs in tissues and cells were isolated using the TRIzol method (Invitrogen) according to the manufacturer’s instructions. In the process of serum RNA extraction, 1 ug lyophilized cel-miR-39-3p (Qiagen, Hilden, Germany) was added as exogenous reference according to the provider recommendations and a miRNeasy serum/plasma kit was used to extract serum cell-free miRNAs according to manufacturer’s protocol. The Spectrophotometer ND-1000 (NanoDrop Technologies, Wilmington, NC, USA) was used to detect RNA concentration. The expression levels of miRNAs were determined using All-in-One miRNA qRT-PCR Detection Kit (GeneCopeia, Rockville, MD, USA) in the ABI 7500 System (Applied Biosystems, Foster City, CA, USA) according to the protocol. The relative expression of miR-371a-5p in tissues and cells were normalized to U6 and that in serum to cel-miR-39 using the 2−ΔΔCt method. The primers were as follows:
cel-miR-39, 5′-CAGAGTCACCGGGTGTAAAT-3′;
miR-371a-5p, 5′-TGCGGACTCAAACTGTGGGGGC-3′;
u6, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′.
Lv Z., Qiu X., Jin P., Li Z., Zhang Y., Lv L, & Song F. (2022). MiR-371a-5p Positively Associates with Hepatocellular Carcinoma Malignancy but Sensitizes Cancer Cells to Oxaliplatin by Suppressing BECN1-Dependent Autophagy. Life, 12(10), 1651.
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Publication 2022
Cells MicroRNAs Oligonucleotide Primers Plasma Serum Tissues trizol
15
miRNA Extraction and Quantification Protocol
The total RNA from tissues and cells was extracted directly using the miRNeasy Mini Kit (QIAGEN, USA). Briefly, tissues or cells were collected in a reaction tube, lysed with 700 µl QIAzol and mixed with 140 µl chloroform. After being centrifuged at 12,000g for 15 min at 4 °C, the upper aqueous phase was transferred to an RNeasy Mini spin column in a 2-ml collection tube and mixed with 100% ethanol. After being washed with 700 µl Buffer RWT and 500 µl Buffer RPE, the total RNA was collected and quantify by NanoDrop 3000 (Life Technologies). For extraction and quantification of miR-338-3p and miR-3065-5p in the collected serum, 1 mL serum was used, and cel-miR-39-3p (hereafter as spike-in, 20 fmol spike-in/200ul serum) was used as spike-in control. Serum were mixed with 500uL of QIAzol and followed by the protocol described above. All miRNA samples were reverse-transcribed and quantified by miRNA Reverse Transcription Kit (QIAGEN) and miScript SYBR Green PCR Kit (QIAGEN). For quantification of miRNA in cells or tissues, U6 was used as control, and for miRNA in serum, spike-in was used as internal control.
Lin C., Yu S., Jin R., Xiao Y., Pan M., Pei F., Zhu X., Huang H., Zhang Z., Chen S., Liu H, & Chen Z. (2019). Circulating miR-338 Cluster activities on osteoblast differentiation: Potential Diagnostic and Therapeutic Targets for Postmenopausal Osteoporosis. Theranostics, 9(13), 3780-3797.
Publication 2019
Buffers Cells Chloroform Ethanol MicroRNAs Reverse Transcription Serum SYBR Green I Tissues
16
Quantification of miR-378 and miR-210 by TaqMan Assay
MiR-378 and miR-210 were quantified by TaqMan MicroRNA Assays (Applied Biosystems, Carlsbad, CA, USA) following reverse transcription (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems) of 3 µL of RNA on Applied Biosystems 7500 instrument following the manufacturers’ protocols. To reduce the possible high intra-assay variance introduced by low abundant miRNA, a pre-amplification step using TaqMan PreAmp Master Mix (Applied Biosystems) was performed for serum RNA samples prior to miR-210 analysis according to the manufacturer’s instructions. Absolute quantification of miRNAs was performed in triplicate.
Fedorko M., Stanik M., Iliev R., Redova-Lojova M., Machackova T., Svoboda M., Pacik D., Dolezel J, & Slaby O. (2015). Combination of MiR-378 and MiR-210 Serum Levels Enables Sensitive Detection of Renal Cell Carcinoma. International Journal of Molecular Sciences, 16(10), 23382-23389.
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Publication 2015
Biological Assay MicroRNAs Reverse Transcription Serum
17
Quantitative miRNA Expression Analysis
MiRNAs were measured using Taqman miRNA assay kits (Applied Biosystems, USA) according to the manufacturer's protocol. Briefly, about 30 ng enriched RNA was reverse transcribed with a TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, USA) in a 15 µL reaction volume. Expression levels of miR-21 and miR-152 were quantified in triplicate by qRT-PCR using human TaqMan MicroRNA Assay Kits (Applied Biosystems, USA) on Eppendorf iplex 4 system (Eppendorf North America, Hauppauge, NY). Spiked-in Cel-miR-39 was used as a normalizer for plasma miRNA quantification.
Chen H., Liu H., Zou H., Chen R., Dou Y., Sheng S., Dai S., Ai J., Melson J., Kittles R.A., Pirooznia M., Liptay M.J., Borgia J.A, & Deng Y. (2016). Evaluation of Plasma miR-21 and miR-152 as Diagnostic Biomarkers for Common Types of Human Cancers. Journal of Cancer, 7(5), 490-499.
Publication 2016
Biological Assay Homo sapiens Iplex MicroRNAs Plasma Reverse Transcription
18
Quantification of Circulating miRNAs
RNA was extracted from 800 µl plasma using an miRNeasy serum/plasma kit (Qiagen China, Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The RNA was eluted in 50 µl nuclease free water and 40 ng RNA (in 5 µl) was reverse transcribed with TaqMan® MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. cDNA was quantitated using a TaqMan-based RT-qPCR assay specific for miR-208a and miR-370 according to the manufacturer's recommended protocol on an Applied Biosystems 7500 Sequence Detection System (the miR-208a and miR-370 specific primers for RT and PCR were enclosed in the kits). The cycling conditions were 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. All reactions were performed in triplicate. The expression levels of circulating miRNAs are presented as the quantitation cycle (Cq) values. Exogenously added cel-miR-39 served as a control for normalizing the data. The comparative Cq (ΔCq) method was used to analyze the miRNA expression levels (15 (link)).
Liu H., Yang N., Fei Z., Qiu J., Ma D., Liu X., Cai G, & Li S. (2016). Analysis of plasma miR-208a and miR-370 expression levels for early diagnosis of coronary artery disease. Biomedical Reports, 5(3), 332-336.
Publication 2016
Biological Assay Circulating MicroRNA DNA, Complementary MicroRNAs Oligonucleotide Primers Plasma Reverse Transcription Serum
19
Melanocyte Growth Supplement Protocol
Medium 254 and human melanocyte growth supplement were purchased from Cascade Biologics (Portland, OR). Dulbecco modified Eagle medium, penicillin‐streptomycin and 0.5% trypsin‐ethylene diamine tetra acetic acid were obtained from GIBCO Invitrogen (Auckland, NZ). The primary and secondary antibodies used in this study were purchased from Santa Cruz Biotech (Santa Cruz, CA), Cell Signaling Technology (Danver, MA), or BD Biosciences (San Jose, CA). Chemiluminescence detection kits were purchased from GE Healthcare (Piscataway, NJ). All other chemicals, including α‐MSH, were purchased from Sigma‐Aldrich (St Louis, MO).
Kim J.H., Kim D.H., Cho K.M., Kim K.H, & Kang N.J. (2018). Effect of 3,6‐anhydro‐l‐galactose on α‐melanocyte stimulating hormone‐induced melanogenesis in human melanocytes and a skin‐equivalent model. Journal of Cellular Biochemistry, 119(9), 7643-7656.
Publication 2018
Acetic Acid alpha Melanocyte Stimulating Hormone Antibodies Biological Factors Chemiluminescence Dietary Supplements Eagle Ethylenediamines Homo sapiens Melanocyte Penicillins Streptomycin Tetragonopterus Trypsin
20
Plasma miRNA Extraction and Quantification
miRNA from 200 μl plasma was extracted using the miRNeasy Serum/Plasma Kit, according to the manufacturer's recommendations (Qiagen, Germany). Briefly, samples were supplemented (after addition of QIAzol) with 3.5 μl miRNeasy Serum/Plasma Spike-In Control (1.6 × 108 copies/μl working solution; Qiagen, Germany). The cel-miR-39 could be used for normalization of the RNA preparation. The amount and purity of RNA was estimated by quawell micro volume spectrophotometer (Quawell, USA). Subsequently, miRNA was transcribed to cDNA using the miScript II RT Kit (Qiagen, Germany). The cDNA were used for detecting miRNA expression by Q-PCR using the miScript SYBR Green PCR Kit with miScript Primer Assay (Qiagen, Germany). The relative expression level of miRNA was determined by the cycle number via Q-PCR, with levels normalized to the average of cel-miR-39 using the 2−ΔΔCT method.
Ling S., Zhong G., Sun W., Liang F., Wu F., Li H., Li Y., Zhao D., Song J., Jin X., Wu X., Song H., Li Q., Li Y., Chen S., Xiong J, & Li Y. (2017). Circulating microRNAs Correlated with Bone Loss Induced by 45 Days of Bed Rest. Frontiers in Physiology, 8, 69.
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Publication 2017
Biological Assay DNA, Complementary MicroRNAs Oligonucleotide Primers Plasma Serum SYBR Green I
21
Extracellular miRNA Extraction and Purification
Cellular and intravesicular miRNAs were extracted from centrifugation pellets or immunoprecipitates. These were lysed with 700 µL of Qiazol (Qiagen, Venlo, The Netherlands). Extraction was then performed using the “miRNEasy mini kit” (Qiagen) according to the supplier’s recommendations. Eluates were stored at −80 °C before analysis.
The miRNAs from culture supernatants, centrifugation supernatants or filtrates were extracted from 200 µL of the sample. The extraction was then performed using the miRNEasy Advanced Serum/Plasma Kit (Qiagen) according to the supplier’s recommendations.
Extractions from the different kits were performed on the Qiacube automated system (Qiagen).
Cel-miR-39-3p spike-in control, obtained from Qiagen, was added to the samples to either check the quality of the purification or normalize the bkv-miR-B1-5p qPCR results after successive filtrations and or centrifugations.
Demey B., Bentz M., Descamps V., Morel V., Francois C., Castelain S., Helle F, & Brochot E. (2022). BK Polyomavirus bkv-miR-B1-5p: A Stable Micro-RNA to Monitor Active Viral Replication after Kidney Transplantation. International Journal of Molecular Sciences, 23(13), 7240.
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Publication 2022
Cells Centrifugation Filtration MicroRNAs Pellets, Drug Plasma Serum
22
DPPH Radical Scavenging Assay
Antioxidants scavenge the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical by donating a proton, forming the reduced DPPH, and were evaluated by using the methodology [21 (link)]. Various concentrations (50, 75, 100, and 150 µg/mL) of the sample (4.0 mL) were mixed with 1.0 mL of a solution containing the DPPH radical (Sigma Aldrich, Bangalore, India), resulting in the final concentration of DPPH being 0.2 mM. The mixture was shaken vigorously and left to stand for 30 min, and the absorbance was measured at 517 nm. Ascorbic acid (20% Sigma Aldrich, Bangalore, India) was used as a positive control.
Prakash P., Kumari N., Gayathiri E., Selvam K., Ragunathan M.G., Chandrasekaran M., Al-Dosary M.A., Hatamleh A.A., Nadda A.K, & Kumar M. (2021). In Vitro and In Silico Toxicological Properties of Natural Antioxidant Therapeutic Agent Azima tetracantha. LAM. Antioxidants, 10(8), 1307.
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Publication 2021
Antioxidants Ascorbic Acid diphenyl Protons
23
Binding Kinetics of CD137 Antibody
Not available on PMC !

Example 7

Jurkat cells (i.e., an immortalized human T lymphocyte cell line) characterized by stable, over-expression of hCD137 were plated at 20,000 cells/well and stained with a titration of primary murine anti-CD137 antibody BBK2 (BBK2-mlgG1) for 4 hours at 4° C. Secondary anti-mouse AF488 stain, at a constant amount, was added for 30 minutes at 4° C. After washing, plates were run on a flow cytometer and binding of BBK2-mlgG1 (and the negative control, i.e., mlgG1) was determined based on geometric mean fluorescence intensity in the AF488 channel. Results from these assays are provided in FIGS. 1A and B.

As shown in FIGS. 1A and 1B, the murine BBK2 antibody binds to human T cells (i.e. CD137-expressing Jurkat cells), with an EC50=35 pM.

US10434185B2. Compositions and methods for the depletion of CD137+ cells (2019-10-08). Magenta Therapeutics, Inc. [US]. Inventors: Adam Hartigan [US], Anthony Boitano [US], Michael Cooke [US], Megan D. Hoban [US], Rahul Palchaudhuri [US].
Full text: Click here
Patent 2019
Antibodies, Anti-Idiotypic Biological Assay Cell Lines Cells Figs Fluorescence Homo sapiens Immunoglobulins Jurkat Cells Mus Stains T-Lymphocyte Titrimetry TNFRSF9 protein, human
24
Binding Affinity of Anti-CD137 Antibody
Not available on PMC !

Example 7

Jurkat cells (i.e., an immortalized human T lymphocyte cell line) characterized by stable, over-expression of hCD137 were plated at 20,000 cells/well and stained with a titration of primary murine anti-CD137 antibody BBK2 (BBK2-mIgG1) for 4 hours at 4° C. Secondary anti-mouse AF488 stain, at a constant amount, was added for 30 minutes at 4° C. After washing, plates were run on a flow cytometer and binding of BBK2-mIgG1 (and the negative control, i.e., mIgG1) was determined based on geometric mean fluorescence intensity in the AF488 channel. Results from these assays are provided in FIGS. 1A and B.

As shown in FIGS. 1A and 1B, the murine BBK2 antibody binds to human T cells (i.e. CD137-expressing Jurkat cells), with an EC50=35 pM.

US10576161B2. Compositions and methods for the depletion of CD137+ cells (2020-03-03). Magenta Therapeutics, Inc. [US]. Inventors: Adam Hartigan [US], Anthony Boitano [US], Michael Cooke [US], Megan D. Hoban [US], Rahul Palchaudhuri [US].
Full text: Click here
Patent 2020
Antibodies, Anti-Idiotypic Biological Assay Cell Lines Cells Figs Fluorescence Homo sapiens Immunoglobulins Jurkat Cells Mus Stains T-Lymphocyte Titrimetry TNFRSF9 protein, human
25
Anti-CD137 Antibody Binding Assay
Not available on PMC !

Example 3

This example illustrates binding of anti-CD137 antibody to Jurkat cells.

Purified anti-CD137 antibody leads were also applied to CD137-induced Jurkat cells to determine binding activity by FACS. Jurkat cells were treated with PMA (10 ng/ml) and ionomycin (1 μg/ml) to induce CD137 expression for 2 days. Stimulated cells were incubated with anti-CD137 (0.5 μg/ml) and reference (ref) Ab (0.5 μg/ml) as positive control for 1 hr on ice, left unstained or incubated with OX40 reference (ref) Ab as negative controls. The cells were washed three times with 1×PBS and then incubated with Alexa-488-conjugated goat anti-human IgG (H+L) (Invitrogen Inc.) on ice for an additional 1 hr. After staining, the cells were washed three times with 1×PBS, resuspended in 1×PBS before being analyzed by FACS Calibur (BD Biosciences, Inc.) and FlowJo (TreeStar, LLC). Among CD137 antibody leads, several leads possessed binding activity comparable to the reference antibody, as shown in FIG. 4.

These results show binding of anti-CD137 antibody leads to activation of Jurkat cells, as seen by flow cytometry.

US11725058B2. CD137 antibodies for T-cell activation (2023-08-15). AP Biosciences, Inc. [TW]. Inventors: Jhong-Jhe You [TW], Ching-Hsuan Hsu [TW], Po-Lin Huang [TW], Jeng-Horng Her [US].
Full text: Click here
Patent 2023
anti-IgG Antibodies Antibodies, Anti-Idiotypic Cells Flow Cytometry Goat Homo sapiens Immunoglobulins Ionomycin Jurkat Cells T-Lymphocyte TNFRSF4 protein, human TNFRSF9 protein, human Vision
26
Binding Capability of Anti-OX40 Antibodies
Not available on PMC !

Example 1

Experiments were performed to determine the binding capability of various anti-OX40 antibodies. Clones from the anti-OX40 antibody library were tested for binding on anti-CD3/anti-CD28 pre-activated human peripheral blood mononuclear cell (PBMC) for 3 days. Each clone was incubated at 5 μg/ml with 3×105 pre-activated human PBMCs. The binding of anti-OX40 antibodies to activated human T cells was revealed by flow cytometry using an APC-labelled anti-human Fc secondary antibody. A phycoerythrin (PE)-labelled anti-hCD3 antibody was also used to show that the fluorescence was detected on the T-cells. Secondary antibody alone was used as a negative control. A commercially available APC-labelled anti-human OX40 (clone Ber-ACT35) was used as positive control. The results are shown in FIG. 1. FIG. 1 shows the binding of anti-OX40 antibody clones on activated human T-cells measured by the percent CD3+OX40+ T cells. The anti-OX40 clones that were tested are shown on the x-axis. FIG. 1 shows that most of the tested anti-OX40 clones are bound on T cells.

US11623962B2. Anti-OX40 binding proteins (2023-04-11). Sorrento Therapeutics, Inc. [US]. Inventors: Damien Bresson [US].
Full text: Click here
Patent 2023
Anti-Antibodies Antibodies, Anti-Idiotypic cDNA Library Clone Cells Epistropheus Flow Cytometry Fluorescence Homo sapiens Immunoglobulin Fc Fragments Immunoglobulins Muromonab-CD3 OX40 Receptors PBMC Peripheral Blood Mononuclear Cells Phycoerythrin T-Lymphocyte TNFRSF4 protein, human
27
Cell Binding Affinity of VSIG8-Fc-OX40L
Not available on PMC !

Example 4

Cell binding assays were performed to demonstrate the binding affinity of the different domains of the human VSIG8-Fc-OX40L chimeric protein towards their respective binding partners on the surface of a mammalian cell membrane.

For cell binding assays, immortalized cell lines were engineered to stably express human OX40 (Jurkat/hOX40). Increasing concentrations of the VSIG8-Fc-OX40L chimeric protein were incubated with the over-expressing (Jurkat/hOX40) cell line for two hours. Cells were collected, washed, and stained with antibodies for the detection of chimeric protein binding by flow cytometry.

As shown in FIG. 4, the VSIG8-Fc-OX40L chimeric protein bound to OX40 present on the cell surface in a concentration-dependent manner and with low nM affinity. Specifically, as shown in FIG. 4, the cell binding assay demonstrated that VSIG8-Fc-OX40L binds to OX40 with an affinity of about 30 nM (according to the EC50 calculation).

US11834488B2. VSIG8-based chimeric proteins (2023-12-05). Shattuck Labs, Inc. [US]. Inventors: Taylor Schreiber [US], George Fromm [US], Suresh De Silva [US].
Full text: Click here
Patent 2023
Antibodies Binding Proteins Biological Assay Cell Lines Cells Chimera Flow Cytometry Homo sapiens Mammals Plasma Membrane TNFRSF4 protein, human TNFSF4 protein, human Tumor Necrosis Factor Ligand Superfamily Member 4
28
Characterizing T Cell Activation Reporters
The mouse thymoma cell line Bw5417 (short designation within this work Bw) and Jurkat E6.1 (JE6.1), were cultured as described (45 (link)). Triple parameter reporter cell lines (TPR) and the monoreporter cell line are based on the JE6.1 Jurkat cell line, stably expressing NFκB::eCFP, NFAT::eGFP, and AP-1::mCherry reporter constructs or NFκB::eGFP, respectively as described (46 (link)).
T cell stimulator cells (TCS) used in this study are Bw5147 cells that stably express membrane-bound single chain antibody fragments derived from the CD3 antibodies (mb-α-CD3) UCHT1 or OKT3 on their surface (47 (link), 48 (link)).
A CD14 mAb antibody was used to stain the surface expression of aCD3scFv which were expressed on the cell surface via a c-terminal CD14 sequence (49 (link)). To exclude the TCS in the reporter assays, an mCD45 antibody was used.
The following flow cytometry antibodies were used in this study: PE-Isotype control (MPOC-21), PE-41BBL (5F4), PE-CD70 (113–16), PE-OX40L (11C3.1), PE-41BB (CD137, 4B4-1), PE-CD27(M-T271), PE-GITR (621), PE-OX40 (CD134, ACT35), APC-CD16 (3G8), APC-CD32 (FUN2), APC-CD64 (10.1), APC-mCD45 (104), APC-CD14 (63D3), PE-CD14 (63D3), FITC-CD56 (HCD56), BV421-CD19 (HIB19), BV421-CD4 (OKT4), PerCP-CD8 (HIT8a, all from Biolegend, San Diego, CA, USA), and PE-GITRL (REA841, Miltenyi Biotec).
For CFSE proliferation assays a functional grade CD3 mAb (UCHT1, Biolegend) was used. For annexin V assays, an FcR silenced CD3 mAb (REA613, Miltenyi Biotec) was used. Agonistic 41BB antibodies - Urelumab (BMS-663513), Utomilumab (PF-05082566), and the CD27 agonist mAb Varlilumab (CDX-1127) were purchased from Creative Biolabs (NY, USA).
For blocking of Fc receptors, cells were incubated for 20 minutes at 4°C with 20 mg/ml Beriglobin (CSL Behring). Flow cytometry analysis was performed using FACSCalibur™ or LSRFortessa™ flow cytometers (BD Bioscience, Franklin Lakes, NJ). FlowJo software (version 10.4.1. Tree Star, Ashland, OR) was used for flow cytometry data analysis.
Leitner J., Egerer R., Waidhofer-Söllner P., Grabmeier-Pfistershammer K, & Steinberger P. (2023). FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab. Frontiers in Immunology, 14, 1208631.
Full text: Click here
Publication 2023
5-(6)-carboxyfluorescein diacetate succinimidyl ester agonists Annexin A5 Antibodies Biological Assay Cardiac Arrest CDX-1127 Cell Lines Cells Fc Receptor Flow Cytometry Fluorescein-5-isothiocyanate Immunoglobulin Isotypes Immunoglobulins Muromonab-CD3 Mus PF-05082566 Receptors, Antigen, B-Cell RELA protein, human Stains T-Lymphocyte Thymoma TNFRSF4 protein, human TNFRSF9 protein, human TNFRSF18 protein, human TNFSF4 protein, human Trees urelumab utomilumab varlilumab
29
Optimized Quantification of Flavonoid Standards
The quantitative analysis of flavonoid standards—fisetin and quercetin (Sigma-Aldrich, USA) was optimized using a lab solution software and an HPLC system of LC2030 (Shimadzu, Japan) on the C18 column (Luna Omega 3 um Polar C18 100, 250 × 4.6 mm, Phenomenex, USA) with slight modification [25 , 26 ]. The solvents used for mobile phase A and phase B were HPLC grade water (Thermo Fisher Scientific, India) with pH-2.15 and acetonitrile respectively. The solvents were degassed for 30 min using a sonicator (Faithful Instrument, China). The gradient elution condition was set at 5 min, 35% B; at 10 min, 40% B; at 15 min, 50% B; and stopped at 20 min. The flow rate was 0.5 ml/min with an autosampler injection volume of 20 µl. The column temperature was 40 °C, and the UV–visible detector was set at 340 nm.
Majhi R., Maharjan R., Shrestha M., Mali A., Basnet A., Baral M., Duwal R., Manandhar R, & Rajbhandari P. (2023). Effect of altitude and solvent on Psidium guajava Linn. leaves extracts: phytochemical analysis, antioxidant, cytotoxicity and antimicrobial activity against food spoilage microbes. BMC Chemistry, 17(1), 36.
Full text: Click here
Publication 2023
acetonitrile fisetin Flavonoids High-Performance Liquid Chromatographies Omega-3 Fatty Acids Quercetin Solvents
30
Genetic Polymorphism Analysis Protocol
5 mL of the venous blood was collected into plain (coagulated) and EDTA (anticoaggulated) tubes. Plain vacutainer consist of 3 mL of the serum sample was used to analyze the biochemical parameters and 2 mL of the EDTA sample was used for molecular analysis. Genomic DNA was extracted from peripheral blood leukocytes using Norgen DNA extraction kit (Norgen Biotek corp, Canada). DNA samples were stored at -80°C. The rs1799883 polymorphism was genotyped using a TaqMan® SNP genotyping assay (Assay ID: C_2834835_10) on a 7300HT sequence detection system (Applied Biosystems, USA). Primers and probes were obtained from Applied Biosystems as Assays-by-Design™. Cases and controls were ensured to have even treatment during the assay procedure, and each plate included negative controls (with no DNA). Plates were read on the ABI Prism 7300 using the Sequence Detection Software (Applied Biosystems) using 40 PCR cycles (92°C denaturation for 15 seconds, 60°C annealing/extension for 60 seconds). Measurements were repeated for samples with failed genotypes. Assays that did not show >95% concordance were discarded and replaced with alternative assays with the same tagging properties.
Alharbi K.K., Khan I.A., Bazzi M.D., Al-Daghri N.M., Hasan T.N., Alnbaheen M.S., Alharbi F.K., Al-Sheikh Y.A., Syed R, & Aboul-Soud M.A. (2014). A54T polymorphism in the fatty acid binding protein 2 studies in a Saudi population with type 2 diabetes mellitus. Lipids in Health and Disease, 13, 61.
Full text: Click here
Publication 2014
Biological Assay BLOOD Edetic Acid Genetic Polymorphism Genome Genotype Leukocytes Neoplasm Metastasis Oligonucleotide Primers prisma Serum Veins

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