Whole blood was obtained from 3 healthy male donors age 20–38 in BD Vacutainer K2 EDTA blood collection tubes. Mononuclear cells were isolated via density gradient-based centrifugation using Ficoll Paque at 1500 RPM for 30 minutes using minimum acceleration and no brake. From roughly 30 mL of whole blood we extracted 25–30 million PBMCs. Isolated mononuclear cells were counted and re-suspended in PBS to a density of 10×106 or 20×106 cells per 100 µL PBS. Scaffold bearing or scaffold and tumour bearing mice were warmed under a heat lamp to dilate the tail vein, placed in a restrainer, and intravenously delivered by tail vein injection using a 27 Gauge needle.
Vacutainer k2 edta blood collection tube
Manufactured by BD
Sourced in United States
The BD Vacutainer® K2 EDTA blood collection tubes are designed for the collection and transportation of blood samples. These tubes contain the anticoagulant K2 EDTA, which prevents blood clotting during the collection process.
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9 protocols using vacutainer k2 edta blood collection tube
1
Isolation and Delivery of Mouse PBMCs
2
Isolation and Delivery of Mouse PBMCs
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Whole blood was obtained from 3 healthy male donors age 20–38 in BD Vacutainer K2 EDTA blood collection tubes. Mononuclear cells were isolated via density gradient-based centrifugation using Ficoll Paque at 1500 RPM for 30 minutes using minimum acceleration and no brake. From roughly 30 mL of whole blood we extracted 25–30 million PBMCs. Isolated mononuclear cells were counted and re-suspended in PBS to a density of 10×106 or 20×106 cells per 100 µL PBS. Scaffold bearing or scaffold and tumour bearing mice were warmed under a heat lamp to dilate the tail vein, placed in a restrainer, and intravenously delivered by tail vein injection using a 27 Gauge needle.
3
Quantifying Intratumoral NPTX Uptake
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Three days after the last injection of DC101 or PBS, NPTX (120 mg·kg−1) or saline control was administered via the tail vein. Two hours after NPTX administration, mice were anesthetized using isoflurane and sacrificed by heart puncture. Blood was collected in BD Vacutainer® K2 EDTA blood collection tubes which were spun down at 1300 RCF for 10 min, and plasma was stored at −80 °C. The tumor was harvested, and pieces of the tumor were either fixed in 4% paraformaldehyde (PFA) or snap‐frozen and stored at −80 °C until further processing. For analysis of intratumoral NPTX uptake following ST DC101, 3 of 5 mice had to be excluded due to failed intravenous NPTX injection in which both plasma and tumor concentrations were too low to analyze. Snap‐frozen tumor samples were processed and measured with liquid chromatography/tandem mass spectrometry (LC‐MS/MS) as described earlier (Steins et al., 2017 ). To correct for injection error, tumor NPTX concentrations were normalized against the plasma NPTX concentration of each mouse (i.e., relative NPTX uptake).
4
Plasma Isolation from Whole Blood
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Samples of 2 × 10 mL of whole blood were collected in BD Vacutainer K2 EDTA blood collection tubes with lavender Hemogard closure or Streck cell-free DNA BCT and were processed within 4 h from collection for BD Vacutainer K2 EDTA tubes and within 72 h for Streck cell-free DNA BCT. For plasma isolation, blood collection tubes were centrifuged at 15 to 25 °C for 10 min at 1600× g using a swing bucket rotor. The isolated plasma (supernatant) was transferred to centrifuge tubes. For plasma samples from BD Vacutainer K2 EDTA tubes, a centrifugation at 15 to 25 °C for 10 min at 3000× g was performed using a fixed angle rotor, while for plasma samples from Streck cell-free DNA BCT, the isolated plasma was centrifuged at 15 to 25 °C for 10 min at 6000× g using a fixed angle rotor. Highly hemolytic samples were rejected. These steps were based on previously published literature and are in line with the Key Recommendations on Assay Characteristics by the United States National Cancer Institute Colon and Rectal–Anal Task Forces [32 (link)].
5
Metastatic Prostate Cancer Blood Samples
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Blood samples from healthy male donors and patients with diagnosed metastatic prostate cancer were provided by the urology service of the “Dr. José Eleuterio González” University Hospital, according to the protocol approved by their Institutional Review Board with number UR16-0007. Prior to blood extraction, informed consent was obtained from healthy donors and cancer patients. Samples were collected in 6 mL BD Vacutainer K2EDTA blood collection tubes (BD, Franklin Lakes, New Jersey, USA) and were processed by our device within 3 hours after the extraction. All procedures involving human participants were performed in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
6
Sampling Protocol for Faeces and Blood in Animals
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Faeces and blood samples were taken from each animal on the first day of the initiation of the ICL system and 70 days after. The sampling was made at 06:30 a.m. while they were still fasting and in their shed. Blood samples were collected from the jugular vein and placed in tubes with EDTA (BD Vacutainer® K2 EDTA Blood Collection Tube; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for haematology analysis and with a vacuum system and gel separator (BD Vacutainer® Blood Collection Tube; Becton, Dickinson and Company, NJ, USA) in others to obtain serum. The vacuum tubes were centrifuged at 1500× g for 5 min and the serum obtained was stored frozen (−80 °C) in Eppendorf tubes (Eppendorf, Hamburg, Germany) until analysis. Faeces were collected directly from the rectum, stored in labelled plastic bags at 4 °C, and taken to the Veterinary Helminthology Laboratory at the UNESP—Institute of Biosciences, Campus of Botucatu, Brazil, where they were analysed.
7
Preoperative Blood Sample Collection
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Blood samples were obtained by jugular vein puncture pre-operatively in tubes with EDTA (BD Vacutainer® K2 EDTA Blood Collection Tube; Becton, Dickinson and Company, USA), to perform the hemogram for every animal. Aliquots of the samples (3 mL) were also placed in plain tubes with gel separators (BD Vacutainer® Blood Collection Tube; Becton, Dickinson and Company, USA) were allowed to clot at room temperature, centrifuged (1,500 × g for 5 min) and the harvest sera were stored in Eppendorf microtubes, stored at −20 °C with biochemical analyses performed within six months.
8
Plasma RNA extraction for viral detection
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EDTA-anticoagulated blood was collected using the BD Vacutainer K2 EDTA blood collection tube. The blood collection tube was centrifuged at 3000× g for 10 min to separate the plasma from the blood cells. After concentration, 400 μL of plasma was subjected to viral RNA extraction using the QIAGEN EZ1 Virus Mini Kit v2.0 (QIAGEN, Hilden, Germany) with the BioRobot EZ1 Advanced XL workstation (QIAGEN) in line with the manufacturer’s protocol. The viral nucleic acid was eluted in 60 μL of elution buffer.
9
Prostate Cancer RALRP Biomarker Study
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