Blood samples were collected by coccygeal venipuncture on the day of embryo collection in experiment 1b (Day 7 after AI), and in experiment 2 (Day 16 after AI) into vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA). The blood samples were left to clot at room temperature for 15 min and stored at 4 °C. The samples were centrifuged at 1500×g for 20 min at 4 °C within an hour of collection and serum was separated and stored at − 20 °C until further utilization. Separate blood samples were collected into vacutainer tubes containing EDTA (Becton Dickinson) to measure PGFM. The blood was centrifuged at 2600×g for 30 min, and the plasma was decanted and frozen at − 20 °C for analysis of the PGFM.
Vacutainer tube
Manufactured by BD
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Vacutainer tubes are laboratory collection tubes used to obtain blood samples from patients. They are designed to maintain the integrity of the collected sample and prevent contamination. The tubes come in various sizes and contain different additives that help preserve the sample for subsequent analysis.
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Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by BD (30 mentions)
K2edta, by BD (10 mentions)
Qiaamp dna blood mini kit, by Qiagen (8 mentions)
Cobas 8000, by Roche (7 mentions)
Histopaque, by Merck Group (7 mentions)
Ficoll paque plus, by GE Healthcare (6 mentions)
Ficoll histopaque, by Merck Group (5 mentions)
Penicillin streptomycin, by Merck Group (5 mentions)
Ficoll hypaque, by GE Healthcare (5 mentions)
Sodium heparin, by BD (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
K3edta, by BD (5 mentions)
Histopaque 1077, by Merck Group (5 mentions)
Acid citrate dextrose solution, by BD (5 mentions)
Leucosep tube, by Greiner (4 mentions)
Facscalibur, by BD (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Elisa kit, by R&D Systems (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
1 110 protocols using vacutainer tube
1
Serum and Plasma Collection for Hormonal Analysis
2
Isolation and Cryopreservation of PBMCs
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About thirty milliliters of whole blood were collected in EDTA-containing vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA). Peripheral blood mononuclear cells (PBMCs) were separated on lymphocyte separation medium (Organon Teknika Corp., Durham, NC, USA), and washed twice in PBS. Leukocytes viability was determined using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA) and cryopreserved at − 80 °C in RPMI 1640 containing 50% fetal bovine serum (FBS) and 10% dimethylsulfoxide (DMSO) until using.
Plasma was obtained from blood collected in EDTA-containing vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA). Samples were prepared by one centrifugation at 2000g for 10 min (no brake) and stored at − 80 °C until testing.
Plasma was obtained from blood collected in EDTA-containing vacutainer tubes (Becton Dickinson and Co., Rutherford, NJ, USA). Samples were prepared by one centrifugation at 2000g for 10 min (no brake) and stored at − 80 °C until testing.
3
Platelet Aggregation Study with Aspirin and Clopidogrel
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After receiving >5 days of aspirin and clopidogrel, blood samples were collected 2 hours after the most recent dose (≈10 am ) into one 2‐mL BD Vacutainer tube (Becton, Dickinson and Company) containing 3.6 mg of K2 EDTA and into two 2‐mL BD Vacutainer tubes containing 0.105 mol/L of buffered sodium citrate (3.2%). Within 1 hour of collection, blood samples were transferred to the central laboratory. EDTA samples were frozen at −80°C for subsequent genotyping, whereas citrated samples were processed immediately for platelet aggregation studies. After centrifuging citrated samples at 200 g for 8 minutes at 22°C, platelet‐rich plasma was carefully separated. The remaining sample was centrifuged at 2465 g for another 10 minutes to obtain platelet‐poor plasma. The platelet count in platelet‐rich plasma was standardized by adding platelet‐poor plasma to achieve a count of 250×109/L. Platelet aggregation tests by light transmission aggregometry were performed within 3 hours of platelet‐rich plasma preparation.10 At 1‐month follow‐up, additional blood samples were collected for repeat platelet aggregation studies.
4
FCoV Antibody Titer Evaluation in Cats
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A total of 2.5 ml blood was collected from FCoV antibody titer ≥ S2+, FIV- and FELV- cats. The collected blood samples were immediately divided into two tubes for different purposes. First, 0.5 ml of blood was stored in clot activator tubes (BD Vacutainer® Tubes with BD hemoguard closure, USA) on ice and kept at 4 °C for serum separation. The remainder of the blood was transferred into EDTA tubes (BD Vacutainer® Tubes with BD hemoguard closure, USA) for PBMC isolation and plasma collection. The collected serum was stored at −80 °C for multiplex bead-based immunoassay. Isolation of PBMC was performed using Ficoll-Paque PLUS (GE Healthcare Life Science, USA) following the steps provided by the manufacturer. Plasma and PBMC were collected separately and stored at −80 °C until further use in real-time PCR for measuring viral load and mRNA expression of immune-related genes.
5
Timing and Processing of CTC Samples
6
Anaerobic Butyl Acetate Synthesis
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To culture the strains for butyl acetate synthesis, 1% (v/v) of overnight cultures in LB was used to inoculate 3 mL TB culture containing 20 g/L glucose with appropriate antibiotics in test tubes. When the cultures reached OD600 of 0.4–0.6, they were switched to anaerobic by transferring them into 10 ml BD vacutainer tubes. Head space was then purged with anaerobic gas (95% N2, 5% H2). When required, 0.3% (v/v) butanol and 1 mM IPTG were added into the cultures at the same time switching to BD vacutainer tubes for butyl acetate synthesis and the induction of protein expression, respectively. Cultures were sampled at specified times for optical density measurement and product quantification.
7
Quantifying Plasma Sulfide Species
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Plasma samples were analyzed for free sulfide, ALS, BSS, and total sulfide levels as we have previously reported [11] (link), [13] (link). Free sulfide was measured using the MBB method as previously reported [11] (link). For detection of ALS and BSS, 50 μl of plasma was added separately into two sets of 4 mL BD vacutainer tubes. Four hundred fifty microliters of 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA) was added to one tube [acid labile reaction] and 450 μl of 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA) plus 1 mM TCEP was added to the second tube [total sulfide reaction]. Following a 30-min incubation on a nutator, the reaction liquid was removed and sulfide gas subsequently trapped by adding 500 μl of 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) into the BD vacutainer tube and incubated again for 30 min on a nutator mixer. The trapping solutions were removed and sulfide levels measured using the MBB method as we have previously reported [13] (link). Determination of ALS was made by reacting plasma samples with acidic phosphate buffer alone and subsequent trapping of evolved sulfide. Measurement of BSS was determined by subtracting the acid labile value from the total sulfide protocol containing TCEP reductant treatment under acidic conditions. Total sulfide levels were directly obtained from the total sulfide reaction.
8
Circulating CXCL12 and MNC Dynamics
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Venous blood was drawn by repeated venepuncture 10min before (baseline) and at several time points after exercise cessation (0, 30, 90, 180, and 270min) from the antecubital vein into 6ml BD vacutainer tubes spray-coated with EDTA anti-coagulant for isolation of MNCs and 2ml BD vacutainer tubes spray-coated with EDTA anti-coagulant for isolation of plasma (Becton Dickinson AG, Allschwil, Switzerland).
Plasma was isolated by immediate centrifugation (1,500g, 10min at room temperature) and stored in aliquots at −80°C until further analysis of CXCL12 concentrations. MNCs were isolated using Ficoll Histopaque-dependent density gradient centrifugation (Ficoll-Paque™_Plus; GE Healthcare, Opfikon, Switzerland). Isolated MNCs were lysed in 1ml peqGOLD TriFast™ (VWR, Dietikon, Switzerland) and stored at −20°C until RNA isolation and subsequent real time PCR (qPCR) analyses.
Plasma was isolated by immediate centrifugation (1,500g, 10min at room temperature) and stored in aliquots at −80°C until further analysis of CXCL12 concentrations. MNCs were isolated using Ficoll Histopaque-dependent density gradient centrifugation (Ficoll-Paque™_Plus; GE Healthcare, Opfikon, Switzerland). Isolated MNCs were lysed in 1ml peqGOLD TriFast™ (VWR, Dietikon, Switzerland) and stored at −20°C until RNA isolation and subsequent real time PCR (qPCR) analyses.
9
Standardized Serum and Plasma Collection
10
Blood Sampling and Processing for Immunological Studies
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Blood samples were collected and processed as previously published [19 (link)]. Briefly, blood (100 mL) was collected from each subject at two different time points: day 0 (baseline before immunization) and day 28 after immunization (peak of humoral immune responses). Each blood sample included: (i) 90 mL of blood collected in 9 BD Vacutainer® tubes containing lithium heparin (anticoagulant) that was used for isolation of peripheral bone mononuclear cells (PBMCs), and (ii) 10 mL of blood collected in 2 BD Vacutainer® tubes (containing clot activator) that was used for the preparation of serum. PBMCs were isolated by gradient centrifugation using Ficoll Paque Plus tube (GE Healthcare Life Sciences, Uppsala, Sweden), following the manufacturer’s instructions. Isolated PBMCs were resuspended in freezing media (20% heat-inactivated FCS, 10% DMSO in RPMI) at 1 × 107 cells/mL and stored in liquid nitrogen for future use. For serum, blood tubes were centrifuged at 2000× g for 15 min at room temperature. Serum was collected from the supernatant after centrifugation and stored at −80 °C.
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