To stimulate BAT thermogenesis, mice were injected with the β3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Ninety min later, mice were anaesthetized and blood was collected by cardiac puncture for plasma acylcarnitine measurements. Blood samples were dried on filter paper (Whatman ProteinSaver 903) and stored at −20 °C. Measurements were done at the Newborn Screening lab at the Children's Hospital of Eastern Ontario (CHEO) using mass spectrometry. Methods are described in [34] .
Protein saver 903
Manufactured by Cytiva
Sourced in United States
Protein Saver 903 is a laboratory equipment designed for the storage and preservation of protein samples. It provides a reliable and efficient way to maintain the integrity of protein samples during storage and transportation.
Automatically generated - may contain errors
Lab products found in correlation
Whatman, by Cytiva (12 mentions)
Acquity ultra performance liquid chromatography system, by Waters Corporation (1 mentions)
Autocad 2016, by Autodesk (1 mentions)
Htx sprayer, by HTX Technologies (1 mentions)
Acquity beh c18, by Waters Corporation (1 mentions)
Vacutainer cpt tube, by BD (1 mentions)
Vacutainer k3edta tubes, by BD (1 mentions)
Ahlstrom 226, by PerkinElmer (1 mentions)
31 et chr, by Cytiva (1 mentions)
3mm chr, by Cytiva (1 mentions)
Thermomixer comfort, by Eppendorf (1 mentions)
Microtube, by Sarstedt (1 mentions)
Pursuit xrs diphenyl, by Agilent Technologies (1 mentions)
Microvette cb 300 k2e, by Sarstedt (1 mentions)
Inno lia, by Fujirebio (1 mentions)
Hiv uni form 2 plus o, by bioMérieux (1 mentions)
Microtainer tube, by BD (1 mentions)
Milli q apparatus, by Merck Group (1 mentions)
12 protocols using protein saver 903
1
β3-Adrenergic Agonist Stimulated BAT Thermogenesis
2
Measurement of Serum Biomarkers and TFV-DP Levels
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Serum 25(OH)D and iPTH levels were measured by chemiluminescent microparticle immunoassay. A 25(OH)D level <20 ng/mL and 20 to 30 ng/mL were defined as vitamin D deficiency and insufficiency respectively [24 ]. The upper normal limit of serum iPTH used was 65 pg/mL [36 ]. Serum for 25(OH)D and iPTH stored at −70°C until analysis. Serum calcium, phosphorus and alkaline phosphatase were measured using the VITROS Ca, VITROS PHOS and VITROS ALKP slide methods respectively.
TFV‐DP concentrations were measured using liquid chromatography mass spectrometry (LC‐MS/MS). Venous blood was collected in EDTA‐K2 tubes and 25 µL was spotted onto Whatman Protein Saver 903 cards (minimum 3 spots/card) and dried for at least three hours. Dried blood spots were stored at −70°C until analysis. For TFV‐DP quantification, a 3mm punch was taken from a single spot using a micro‐puncher. The dried blood was lysed from the paper using methanol: H2O and then extracted and purified using Solid Phase Extraction (SPE). TFV‐DP was eluted through the SPE cartridge and then dephosphorylated using alkaline phosphatase. The resulting tenofovir (TFV) was then purified and eluted via SPE and the samples analysed using LC‐MS/MS. The TFV‐DP calibration curve range was 200 to 10 000 fmol/3 mm punch [37 ].
TFV‐DP concentrations were measured using liquid chromatography mass spectrometry (LC‐MS/MS). Venous blood was collected in EDTA‐K2 tubes and 25 µL was spotted onto Whatman Protein Saver 903 cards (minimum 3 spots/card) and dried for at least three hours. Dried blood spots were stored at −70°C until analysis. For TFV‐DP quantification, a 3mm punch was taken from a single spot using a micro‐puncher. The dried blood was lysed from the paper using methanol: H2O and then extracted and purified using Solid Phase Extraction (SPE). TFV‐DP was eluted through the SPE cartridge and then dephosphorylated using alkaline phosphatase. The resulting tenofovir (TFV) was then purified and eluted via SPE and the samples analysed using LC‐MS/MS. The TFV‐DP calibration curve range was 200 to 10 000 fmol/3 mm punch [
3
DBS Analysis Using UPLC-FLD and MALDI-Orbitrap
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The DBS cards used were Protein saver 903 of Whatman (Florham Park, NJ, USA). A regular office puncher with a diameter of 5.9 mm, and a 1.2-mm puncher purchased from Harris (CA, USA) were used to collect the samples. Analyses were performed on an Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters, Milford, CT, USA) coupled to a FLD detector. The chromatographic column used was an Acquity BEH C18 (2.1 × 50 mm, 1.7 µm) from Waters. System control, data collection, and data processing were accomplished using Empower 2 software (version 6.20) from Waters. The statistical treatment of the results was carried out using Microsoft Excel 2010 (Redmond, Washington, DC, USA). AutoCAD2016 software (AUTODESK, San Rafael, CA, USA) was used for measuring the surface of the blood drops in the DBS. The pH was measured with a Crison GPL22 pH-meter (Barcelona, Spain).
For MALDI (Matrix Assisted Laser Desorption) analysis, a MALDI LTQ 25 Orbitrap XL (ThermoFisher, CA, USA), equipped with an N2 laser (LTB, Berlin, Germany, model MNL 100, 100 μJ max power, elliptical spot, 60 Hz repetition rate), was used. The matrix was deposited with the aid of a HTX TM-Sprayer (HTX Technologies, NC, USA). Data treatment was performed using Thermo ImageQuest.
For MALDI (Matrix Assisted Laser Desorption) analysis, a MALDI LTQ 25 Orbitrap XL (ThermoFisher, CA, USA), equipped with an N2 laser (LTB, Berlin, Germany, model MNL 100, 100 μJ max power, elliptical spot, 60 Hz repetition rate), was used. The matrix was deposited with the aid of a HTX TM-Sprayer (HTX Technologies, NC, USA). Data treatment was performed using Thermo ImageQuest.
4
VLCADD Newborn Screening using DBS
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DBS cards used in this study were collected from the metabolomics section in the Center for Genomic Medicine at KFSHRC. Thirty DBS cards included in this study were collected from genetically and biochemically confirmed VLCADD newborns (n = 15) and healthy controls (n = 15). These healthy controls were age- and gender-matched with the patient group (Scheme 1 ). The inclusion criteria of this study were applied to the following cases. Firstly, VLCADD patients were only diagnosed with VLCADD. Secondly, the age of participants was a month at maximum. Any study participants not fitting the inclusion criteria were excluded from the study. DBS cards were prepared by dripping blood samples collected from VLCADD and healthy newborns on filter paper called Whatman ProteinSaver 903 using the heel prick method. After that, the DBS cards were dried before storing them in a sealed bag at 4 °C, pending further metabolomics analysis.
5
SARS-CoV-2 Antibody Detection from Dried Blood Spots
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Upon receipt of boxes, the sample cards (Whatman® Protein Saver 903) were heat-treated (30 minutes at 56° C); a single blood spot per card was punched (0.25-inch diameter) and transferred to an ELISA plate. Plates were coated with 1 μg/ml of purified RBD diluted in PBS overnight at 4C and blocked with Tris-Buffered Saline with 0.1% Tween 20 (TBST) containing 5% non-fat dry milk. DBS were eluted in 500 μl of TBST overnight at 4C and 50 μl of each sample was added to the ELISA plate preloaded with 50 μl of TBST containing 2% non-fat dry milk. Samples were then assayed for SARS-CoV-2 antibodies according to published protocols.18 (link),19 The RBD protein was produced in-house via transfection of HEK293T cells using polyethylenimine (Plasmid was a generous gift from Pr. F. Kramer mount Sinai School of Medicine). Batches were control for purity by SDS-PAGE followed by Coomassie staining and ELISA using an anti His-tag monoclonal antibody. Optical densities were read at 405 nm, and each 96-well plate contained seven negative controls and one positive control (serum from PCR-confirmed case at 1/100 dilution). Samples were tested against IgG and positive samples were confirmed and tested with anti IgM antibodies. Optical density values were normalized to the mean optical density of negative controls daily.
6
Comprehensive Blood Sampling and Processing Protocol
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Dried blood spots on filter paper (Protein Saver 903, Whatman), thick blood smear and venous blood (4 or 8 ml depending on whether donor age was below or above 4 years) were collected on dry season cross-sectional visits and at their first malaria episode of the transmission season. Blood samples drawn by venipuncture were collected in sodium citrate-containing cell preparation tubes (Vacutainer CPT Tubes, BD) and transported to the laboratory where PBMCs, plasma and RBC pellets were separated by centrifugation and used freshly or stored at -80 °C within 3 h. An additional 2 ml of venous blood from RDT + individuals who tested at the end of the dry season (May 2012) cross-sectional 13 , and from individuals at their first malaria episode of the ensuing transmission season, was collected into EDTA tubes (Vacutainer K3EDTA Tubes, BD) and processed directly at the field site. Plasma (used for metabolomic analysis) was separated by centrifugation and immediately frozen in liquid N 2 . The buffy coat was discarded, and leucocytes were removed from the RBC pellet in a two-step procedure: first by density gradient on Lymphoprep solution (Fresenius Kabi), followed by Plasmodipur (EuroProxima) filtration, all according to the manufacturer instructions and as previously described 61 . RBC pellets were then frozen in liquid N 2 and were later used for the RNA-seq and RT-qPCR validation.
7
Quantification of Chloroquine and Metabolite
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Stock solutions (1 mg/ml) of chloroquine, desethylchloroquine and their stable isotope-labeled internal standards were prepared in acetonitrile–water (50–50, v/v) containing 0.5% formic acid and stored at -80°C. Working solutions were prepared from the stock solution using acetonitrile–water (50–50, v/v) as dilution solution and then used for the spiking of whole blood, plasma and whole blood for DBS.
Unless otherwise stated, blank blood from healthy volunteers with EDTA as anticoagulant was used. Plasma was obtained by centrifugation of whole blood at 1500–2000 × g for 10 min [34 ,35 (link)]. Whole blood applied on chromatography filter paper Whatman (31 ET Chr, DMPK-C, 903 Protein saver and 3 MM Chr; Whatman, Buckinghamshire, UK) and an alternative brand, Ahlstrom 226 (PerkinElmer, MA, USA) was used for DBS technique. The calibration curves of chloroquine/desethylchloroquine were 2.56–1220/3.36–1220 ng/ml, 1.41–610/1.41–610 ng/ml and 1.82–1552/2.95–1552 ng/ml in whole blood, plasma and DBS, respectively. The final volume of working solution in blank blood was kept below 5% in all samples.
Unless otherwise stated, blank blood from healthy volunteers with EDTA as anticoagulant was used. Plasma was obtained by centrifugation of whole blood at 1500–2000 × g for 10 min [34 ,35 (link)]. Whole blood applied on chromatography filter paper Whatman (31 ET Chr, DMPK-C, 903 Protein saver and 3 MM Chr; Whatman, Buckinghamshire, UK) and an alternative brand, Ahlstrom 226 (PerkinElmer, MA, USA) was used for DBS technique. The calibration curves of chloroquine/desethylchloroquine were 2.56–1220/3.36–1220 ng/ml, 1.41–610/1.41–610 ng/ml and 1.82–1552/2.95–1552 ng/ml in whole blood, plasma and DBS, respectively. The final volume of working solution in blank blood was kept below 5% in all samples.
8
Microbiome Sample Preparation Protocol
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Microtubes were from Sarstedt, and the thermomixer used was a Thermomixer comfort (Eppendorf). Filter cards were 903 Protein saver and Flinders Technology Associates (FTA) micro cards, both from Whatman (Cytiva) and PerkinElmer 226 cards. The LC column was a Pursuit XRs Diphenyl (Agilent Technologies) (250 × 2.0 mm, particle size 3 µm).
9
Paracheck Pf RDT for Asymptomatic Malaria
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Drops of blood were collected by fingerprick for Paracheck Pf RDT (Orchid Biomedical Systems), which detects the presence of P. falciparum histidine-rich protein 2 in blood specimens; whole-blood ribonucleic acid (RNA); thick and thin blood smears; and dried blood spots (DBS) on filter paper (903 Protein Saver; Whatman). Individuals who tested positive for asymptomatic P. falciparum infection by RDT were treated at the point-of-care using the standard regimen recommended by the Ministry of Health in Kenya. For whole-blood RNA, 200 μl of peripheral fingerprick blood was collected using capillary blood collection tubes containing Tris-ethylenediaminetetraacetic acid (EDTA; Microvette CB300 K2E; Sarstedt) and transferred immediately in cryotubes pre-filled with 400 μl Tempus solution (Applied Biosystems). Filled sample tubes were agitated vigorously per the manufacturer’s instructions and stored at − 80 °C within 24 h of collection until use.
10
Serological Testing for HIV in Burkina Faso
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As part of the Burkina Faso DHS,21 dried blood spot samples (two to five per filter paper; Whatman 903 Protein Saver, Dassel, Germany) obtained by finger prick, were sent to the national blood transfusion centre in individual bags with dessicant and stored at −20 °C. Samples were punched into microtitration plates and analysed for HIV infection using an enzyme-linked immunosorbent assay (Vironostika HIV Uni-Form II plus O, Biomérieux, France). The positive samples and 10% of the negative samples were checked using a recombinant enzyme-linked immunosorbent assay (Enzygnost Anti-HIV 1/2 plus, Dade Behring Marburg GmbH, Germany). All discordant results were tested (InnoLia, Innogenetics, Belgium) once more to give a final result of 160 HIV-positive samples. HIV analysis exhausted a total of 491 samples, leaving 14 886 HIV-negative samples for hepatitis testing.
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