Ethical approval for using whole blood from healthy donor was obtained from Ethics Commission University Clinic of Tuebingen, Germany (349/2013BO2) and experiments were conducted in accordance with relevant guidelines and regulations. Informed consent form (following WHO guideline) was signed by each volunteer (healthy donor) and kept safely by principal investigator for privacy requirement. Blood samples from three healthy donors were collected in EDTA collection tubes (www.sarstedt.com ). For each treatment group, 2 ml of EDTA-blood was transferred into 12-well plates and treated accordingly. R848 (Resiquimod, www.sigmaaldrich.com ) was added at a concentration of 1 mg/ml to the respective blood positive controls. cmRNAhCFTR and pDNAhCFTR (7 picomol each) were complexed to NPs at a ratio of 1:10. The samples were kept at 37 °C incubator maintaining 5% CO2. At 6 h and 24 h, 1 ml of whole blood was transferred into microtubes containing serum gel (www.sarstedt.com ) and spun down at 10,000 × g for 5 min to obtain serum. Sera were stored at −20 °C for further cytokine measurement analyses. Serum was used to conduct IFN-α, TNF-α and IL-8 ELISA at manufacturer’s instruction (www.thermofisher.com ).
Serum gel
Manufactured by Sarstedt
Sourced in Germany
The Serum gel is a laboratory product used to separate serum from whole blood samples. It is a physical barrier that facilitates the separation of the serum layer from the cellular components of the blood during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Edta collection tubes, by Sarstedt (1 mentions)
Procyte dx, by IDEXX (1 mentions)
Nsaid 6 clips, by IDEXX (1 mentions)
Catalyst dx chemistry analyzer, by IDEXX (1 mentions)
S monovette, by Sarstedt (1 mentions)
K3 edta, by Sarstedt (1 mentions)
Li heparin, by Sarstedt (1 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
High binding elisa plates, by Thermo Fisher Scientific (1 mentions)
Nb200 540b, by Novus Biologicals (1 mentions)
Streptavidin hrp, by Mabtech (1 mentions)
Clotting activator, by Sarstedt (1 mentions)
Heparin, by Avantor (1 mentions)
10 protocols using serum gel
1
Cytokine Profiling of CFTR Nanotherapies
2
Diurnal Variation in Serum Biomarkers
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Data collection was carried out between January 2018 and June 2019. Medical history and medication were reviewed by a physician using a standardized questionnaire. Participants were randomly allocated to one of five time slots (08:00, 10:00, 12:00, 14:00 and 16:00) for the measurements, which took around 4 h in total. They were instructed not to diverge from habitual eating behavior (for the previous 72 h), to avoid exercising, drinking alcohol (for the previous 24 h) and drinking caffeinated beverages (for the previous 4 h). On the day of sample collection, participants took the prescribed medication as usual. Fasting blood samples (at least 3 h fasting time) were collected before any kind of measurements involving physical activity. Trained medical staff collected serum samples (2 × 7.5 mL serum-gel, Monovette®, Sarstedt, Nümbrecht, Germany) by venipuncture in the cubital fossa. Serum samples were slightly shaken for 30 min, centrifuged (3,000 rpm; 10 min; 20–23°C) and aliquoted before being frozen at −80°C.
3
Evaluating Tissue Damage Through Blood and Histological Analysis
4
Cytokine and Lipid Mediator Profiling in COVID-19
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For the COVID-19 study, peripheral venous blood was collected in 9 mL K3-EDTA (ethylenediaminetetraacetic acid), 2.7 mL Serum-Gel and 2.6 mL Citrate 3.2% Monovettes (Sarstedt, Nümbrecht, Germany). The Serum-Gel and citrate-stabilized blood samples were centrifuged (10 min, 800 × g, 20 °C) to separate the serum or plasma fraction. Serum (1 mL) samples were stored at −80 °C for cytokine measurement using ELISA. Citrate-plasma samples (1 mL) were immediately frozen and stored at −80 °C for LM profiling. For experimental details on the isolation of PBMCs, monocytes, and neutrophils, see SI Appendix .
5
SARS-CoV-2 RNA Detection and Quantification
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Respiratory tract samples (i.e., tracheal aspirates and nasopharyngeal swabs (eSwab, Copan, Italy)) and blood plasma (Serum-Gel, Sarstedt, Nümbrecht, Germany) were obtained as part of the clinical routine at the Department of Intensive Care Medicine. SARS-CoV-2 RNA was detected and quantified as described previously [18 (link)]. Standard RNA reference material (obtained from INSTAND e.V., Duesseldorf, Germany) was used for quantification.
6
Biomarker Analysis of Blood Samples
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Blood samples were taken by venipuncture and collected using Li-Heparin, serum gel and EDTA-buffered collection monovettes (Sarstedt, Nümbrecht, Germany). TNF-α, LBP, IL-1β, IL-8 and IL-10 concentrations were determined using the chemiluminescence immunoassay method (DPC Biermann Immunoassay Analyzer Immulite 1000). Ferritin and IL-6 concentrations were detected by the electrochemiluminescence immunoassay method “ECLIA” (Roche immunoassay analyzer COBAS 8000 (e 801 module)). CRP was determined by turbidimetric measurement (Cobas c system). Leukocytes and platelets were measured using the Sysmex reference method for enumeration and counting. The measurement was performed by the Institute of Clinical Chemistry, University of Ulm. EDTA anticoagulated blood samples were centrifuged at 2500 g for 10 min and the plasma was stored on average for ∼6 months (SD = 1.69, range = 2–8 months) at minus 80 °C until the final quantification of analytes. KYN was determined using the protocol established by Bizjak et al. (2021) (link).
Concentrations below the limit of detection at t0 (IL-1β < 1.5 pg/ml [10 of 61], IL-6 < 1.5 pg/ml [39 of 49], IL-8 < 2.0 pg/ml [1 of 49], IL-10 < 1.0 pg/ml [20 of 48], CRP <0.6 mg/ml [39 of 61]) were replaced by half of the detection limit. Due to numerous missing values (values below detection limit), CRP and IL-6 could not be evaluated in this study.
Concentrations below the limit of detection at t0 (IL-1β < 1.5 pg/ml [10 of 61], IL-6 < 1.5 pg/ml [39 of 49], IL-8 < 2.0 pg/ml [1 of 49], IL-10 < 1.0 pg/ml [20 of 48], CRP <0.6 mg/ml [39 of 61]) were replaced by half of the detection limit. Due to numerous missing values (values below detection limit), CRP and IL-6 could not be evaluated in this study.
7
Serum Cytokine and Chemokine Profiling
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For blood samples, saphenous bleeds were performed in one of the legs and blood was collected in tubes containing Serum-Gel (Sarstedt Inc, Fisher Scientific, Cat. NC9315741) to allow serum separation by centrifugation (14 000 rpm) for 10 mins using Eppendorf centrifuge 5418R, then serum was used for measuring cytokine and chemokines. Serum samples were stored at –80C after collection.
8
Quantifying Complement Binding via ELISA
9
Blood Sampling and Tissue Collection
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Venous blood samples to a maximum volume of 20 μL were drawn intermittently from tail and facial veins by aseptic technique using 25-21 gauge needles and were collected into heparin (VWR International, Dublin, Ireland)-containing tubes. A terminal blood sample was drawn by cardiac puncture at the time of euthanasia. Serum was collected in micro-tubes with serum gel and clotting activator (Sarstedt, Wexford, Ireland). Plasma and serum samples were prepared by centrifugation at 10,000×g for 10 min. Serum samples were stored at −80 °C and subsequently analysed for biochemical parameters by NationWide Laboratories (Lancashire, UK). Spleen, lungs, kidneys and liver were dissected immediately after euthanasia.
10
OVA-Specific Antibody ELISA in Mice
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Following vaccination, mice were bled at indicated time points for serological analysis. Serum was obtained by centrifuging whole blood collection in micro sample tube with serum gel (SARSTEDT). ELISA plates were coated overnight at 4 °C with 100 μL of 10 μg OVA for detecting OVA-specific antibodies. StartingBlock™ (PBS-T) blocking buffer (Thermo Scientific) was used for blocking and sample dilution. Specific isotypes were identified using HRP-conjugated isotype-specific anti-mouse antibodies obtained from SouthernBiotech, and developed using Pierce TMB substrate kit. ELISA data were analyzed using an endpoint analysis. Serial dilution of pooled positive control samples was used to produce an assay sensitivity curve (Prism: Asymmetric Sigmoidal, 5PL, X is log), and biological samples were compared to that curve to assign a titer (AU) relative to the assay’s lower threshold (calculated as the average value of six blank wells plus 3 times their standard deviation). Groups were then compared by standard statistical testing using Prism statistical analysis software.
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