Uninfected rhesus blood was collected by venipuncture into sodium heparin vacutainers (BD, Franklin Lakes, NJ, USA) and centrifuged at 800 × g for 3 min. Plasma was removed and RBC were washed once and resuspended in iRPMI at 50% hematocrit, and stored at 4oC for up to two weeks. P. knowlesi H strain-infected pRBC were collected from infected rhesus into sodium heparin vacutainers (BD, Franklin Lakes, NJ, USA). After plasma removal by centrifugation, the pRBC were combined with uninfected rhesus RBC in cultures with complete RPMI (cRPMI, which is iRPMI supplemented with 10 mg/L gentamicin, and 1% Albumax II (Life Technologies, Carlsbad, CA, USA)) at 5% hematocrit and 37°C under a 90% N2, 5% CO2, and 5% O2 gas mixture, with media changes once daily, or twice daily if parasitemia was ≥ 4%. Cultures were monitored by microscopy using Giemsa-stained thin films, and maintained at 0.5–10% parasitemia. Total parasitemias and parasite stage distributions were recorded from counts of 5,000 RBC. Cultures were transferred to iRPMI supplemented with 10% (v/v) pooled sera from 2 – 6 animals, for a minimum of three cycles prior to membrane-feeding assays. The three different pools of sera used for this purpose are listed in Table S1 .
Sodium heparin vacutainer
Manufactured by BD
Sourced in United States, France
Sodium heparin Vacutainers are laboratory blood collection tubes used to collect and store blood samples. They contain the anticoagulant sodium heparin to prevent the blood from clotting during the collection and transportation process.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (2 mentions)
Anti human cd14 magnetic beads, by Miltenyi Biotec (2 mentions)
Anti human cd16 magnetic beads, by Miltenyi Biotec (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Gentamicin, by Thermo Fisher Scientific (2 mentions)
Albumax 2, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Edta vacutainer, by BD (2 mentions)
Lithium heparin vacutainer, by BD (1 mentions)
Easysep direct human neutrophil isolation kit, by STEMCELL (1 mentions)
Rosettesep human monocyte enrichment cocktail, by STEMCELL (1 mentions)
Anti cd14 antibody, by BioLegend (1 mentions)
903 protein saver card, by Cytiva (1 mentions)
Mouse igg2b, by R&D Systems (1 mentions)
Fluorochrome conjugated antibody, by BD (1 mentions)
Ack lysis buffer, by Quality Biological (1 mentions)
Macsquant, by Miltenyi Biotec (1 mentions)
Vacutainer sst, by BD (1 mentions)
44 protocols using sodium heparin vacutainer
1
P. knowlesi Culture from Infected Rhesus Blood
2
Equine Blood Parameters During Exercise
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Blood samples were taken from the jugular vein, alternating sides. Seven jugular blood samples were collected for each horse. The first blood sample was taken directly after the first standardized warm-up, then directly after jumping and 60 min after jumping. Sample four was drawn after the second standardized warm-up. For the track standardized exercise test, blood samples were taken immediately after the 5 m/s and 8 m/s canters and 11 m/s gallops respectively. Rectal temperatures were recorded at the time of each blood sampling and temperature-corrected blood data is reported for pH, and pCO2 levels (Table 1 ).
A sodium heparin Vacutainer (BD) and an EDTA Vacutainer (BD) were filled at each sample. All sodium heparin Vacutainer (BD) samples were analysed trackside. A handheld system (iSTAT, Heska) using CG4+ cartridges (iSTAT, Heska) and corrected for rectal temperature provided blood pH, pCO2 and bicarbonate concentrations. Blood lactate concentrations were measured using another handheld analyser (Lactate Pro, Arkray). Samples were stored on ice. Packed cell volume (PCV, l/l) and total protein concentration (TP, g/L) were measured within twelve hours of collection.
A sodium heparin Vacutainer (BD) and an EDTA Vacutainer (BD) were filled at each sample. All sodium heparin Vacutainer (BD) samples were analysed trackside. A handheld system (iSTAT, Heska) using CG4+ cartridges (iSTAT, Heska) and corrected for rectal temperature provided blood pH, pCO2 and bicarbonate concentrations. Blood lactate concentrations were measured using another handheld analyser (Lactate Pro, Arkray). Samples were stored on ice. Packed cell volume (PCV, l/l) and total protein concentration (TP, g/L) were measured within twelve hours of collection.
3
Isolation of Immune Cells from Blood
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Neutrophils were isolated from fresh venous blood collected in lithium heparin vacutainers (BD, Scoresby, Australia) using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Technologies, Tullamarine, Australia) as per manufacturer’s instructions. Neutrophil purity was assessed by cell morphology using light microscopy of Giemsa stained smears of the isolated cells, and viability was assessed using trypan blue exclusion. Monocytes were isolated from both fresh venous blood, collected in lithium heparin vacutainers (BD), as well as from buffy coats supplied from the Australian Red Cross Blood Service. Monocytes were isolated by negative selection using the RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions. Monocyte purity was assessed by staining (anti-CD14 antibody, BioLegend, San Diego, CA) and measuring CD14+ cells by flow cytometry. Monocytes were either frozen in fetal bovine serum (FBS) in 20% dimethyl sulfoxide (DMSO) in liquid nitrogen for later use or used immediately after isolation. Natural killer (NK) cells were isolated from fresh venous blood collected in sodium heparin vacutainers (BD). NK cells were isolated by negative selection using the RosetteSep Human NK Enrichment Cocktail (STEMCELL Technologies) as per manufacturer’s instructions.
4
Remote Dried Blood Spot Sampling for T-Cell Assay
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Dried blood spot sampling was undertaken remotely, avoiding the need for phlebotomist time and hospital visits by potentially immunosuppressed patients. A kit containing instructions on how to collect samples, lancets to obtain capillary blood, a blood spot collection device (PerkinElmer 226 Spot Saver or Whatman 903 Protein Saver card), and a return mail envelope was posted to participants at their home address. Participants were asked to date their dried blood spot test card and return it using first class surface mail as soon as possible. Samples received at the UHW were stored desiccated at −20°C until analysis; those received at QMUL were stored at room temperature for a maximum of a week prior to spot punching and elution, with subsequent storage at −20°C.
T cell responses to SARS‐CoV‐2 in 16 participants with negative humoral response to SARS‐CoV‐2 were measured using a commercially available whole blood assay (ImmunoServ Ltd.), as previously described.5 Briefly, 10 ml venous blood from each patient was collected into sodium heparin vacutainers (BD) and processed in the laboratory within 6 hours of blood draw.
T cell responses to SARS‐CoV‐2 in 16 participants with negative humoral response to SARS‐CoV‐2 were measured using a commercially available whole blood assay (ImmunoServ Ltd.), as previously described.
5
Multiparametric Flow Cytometry of Immune Cells
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Whole blood, collected in sodium-heparin vacutainers (BD, CA), from patients or healthy volunteers was first subjected to red blood cell lysis using ACK lysis buffer (Quality Biological Inc, MD). Cells were then incubated in sterile PBS supplemented with 2% FCS and fluorochrome-conjugated antibodies against CD3, CD4, CD8, CD14, CD19, CD56 (all from BD, CA) and IL-17RC (FAB22691A) and mouse-IgG2B isotype control (both from R&D, MN), for 30 minutes at room temperature in the dark. Cells were washed three times and analysed on a MACSQuant flow cytometer (Miltenyi Biotec, Germany). All flow cytometry data were analysed using FlowJo 7.6 (Treestar, OR).
6
PBMC Isolation and Stimulation
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Venous blood collected in sodium heparin vacutainers (BD Pharmingen) was processed within 4 h of collection. PBMC were isolated by density-gradient centrifugation over Ficoll. Freshly isolated PBMC were stimulated with hkH37Rv and used for RNA extraction (as described below), as well as measurement of IFN-γ release by ELISPOT analysis, whereas remaining PBMC were cryopreserved in temperature-monitored liquid nitrogen tanks.
7
Comprehensive Blood Collection Protocol
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Peripheral blood is collected into EDTA and SST Vacutainers (BD, Franklin Lakes, NJ, USA). Giemsa-stained peripheral blood smear is prepared to evaluate neutrophil and eosinophil counts. In a subset of the study population, additional blood is collected in PAXgene Blood RNA Tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and Sodium Heparin Vacutainers (BD). Blood collected in PAXgene Blood RNA Tubes will be used to study RNA expression profiles, while blood collected in Sodium Heparin Vacutainers will be used for detailed immunological measurements as described below (section Immunological methods). All samples deriving from EDTA and SST Vacutainers (serum, plasma, cell pellet and whole blood) and all PAXgene Blood RNA Tubes are kept at −20°C at the Field Clinical Research Centre (FCRC) and will be sent on dry ice to the University’s laboratory for storage at −80°C.
8
Evaluating Capillary Blood Samples
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Venous blood samples were obtained by venepuncture into 6 or 10 ml lithium or sodium heparin vacutainers (BD). Capillary blood samples were obtained by lancet blade incision of a finger and subsequent collection into a heparin microtainer (BD). A minimum of 400 μl blood was required; any sample underfilled below this amount was rejected. Other reasons for sample rejection included profuse clotting and/or haemolysis and viscous plasma that could not be collected for assays (Supplementary Fig. 5 ). Overall, 299 capillary blood samples were evaluable for antibody responses; of these, 270 samples were also evaluable for T cell responses.
9
Isolation and Stimulation of Human Monocyte-Derived Macrophages
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Venous blood was collected in Sodium-Heparin vacutainers (Cat# 367874, BD). Plasma and blood cells were separated using Histopaque-1077 (Cat# 10771, Sigma) according to the manufacturer’s protocol. Briefly, whole blood was diluted 1:1 with sterile DPBS-2 mM EDTA (Sigma or Corning) and overlaid (30 mL) on to 10 mL of Histopaque-1077. Gradients were centrifuged at 500xg for 30 min. The peripheral blood mononuclear cell (PBMC) layer at the plasma-Histopaque interface was transferred to a new tube and washed twice with cold DBPS-EDTA. PBMCs were isolated and CD16+ cells depleted using anti-human CD16 magnetic beads (Cat# 130-045-701, Miltenyi) using manufacturer’s protocol. After CD16 depletion, CD14+ monocytes were purified using anti-human CD14 magnetic beads (Cat# 130-050-201, Miltenyi). Monocytes were plated in 24-well tissue culture treated plates at density 5x105 per well in 1 mL of cRPMI supplemented with human recombinant M-CSF (50 ng/ml; Peprotech). Every two days the media was exchanges for fresh cRPMI containing M-CSF. At the day 7-8 the media was changed to cRPMI without M-CSF and stimulations were carried out as indicated. MoDMs were stimulated with LPS (100 ng/ml) and ATP (5 mM, 45 min).
10
Maternal Blood Sampling in Pregnant Pigs
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On the final day of the treatments, pregnant pigs were sedated with intramuscular injections of Xylazil-20 and Ketamine (Troy Laboratories Pty Ltd, NSW, Australia) at 0.6 mg/kg and 10 mg/kg liveweight, respectively. Ten mL of maternal blood was collected by jugular venepuncture into sodium heparin vacutainers (BD, North Ryde, NSW, Australia) and the blood was immediately centrifuged at 2000 × g for 10 min at 4 °C for plasma collection. The plasma samples were then aliquoted and stored at -20 °C prior to further analysis. In addition, 1 mL blood was collected via a needle and syringe from the auricular vein in pregnant pigs and immediately loaded into an automatic blood gas analyser (Epoc; Alere, Waltham, MA, USA) for venous blood analysis. Pregnant pigs were then euthanised via intracardiac injections of Lethabarb (pentobarbitone sodium; 162.5 mg/kg liveweight; Virbac Animal Health, NSW, Australia).
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