Blood samples were collected, in the morning following an overnight fast, into EDTA‐coated Vacutainers (BD Biosciences, Franklin Lakes, NJ, USA). Peripheral blood mononuclear cells (PBMCs) were immediately isolated, as described.(12 , 14 ) Briefly, peripheral blood samples were diluted 2:1 in sterile phosphate‐buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA), and gently pipetted onto 10 mL of Ficoll‐Paque PLUS density gradient separation solution (Sigma Aldrich, St. Louis, MO, USA), in a 50‐mL conical tube, ensuring clear layer separation. This was centrifuged at 400g for 40 minutes with brakes off, with the resulting PBMC layer carefully aspirated, without collection of excess serum or Ficoll solution. The PBMCs were washed three times in PBS by centrifugation at 100g for 10 minutes to remove contaminant platelets, before being cryopreserved in 90% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA), 10% dimethyl sulfoxide (DMSO) and stored at −80°C for batch analysis.
Edta coated vacutainer
Manufactured by BD
Sourced in United States
EDTA-coated vacutainers are a type of blood collection tube used for the collection, transportation, and storage of blood samples. The tubes are coated with the anticoagulant Ethylenediaminetetraacetic acid (EDTA), which prevents the blood from clotting. This allows the collected blood to be used for various laboratory analyses.
Automatically generated - may contain errors
Lab products found in correlation
Easysep direct human neutrophil isolation kit, by STEMCELL (2 mentions)
Luna fl dual fluorescence cell counter, by Logos Biosystems (2 mentions)
Buffer el, by Qiagen (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Shandon m1 embedding matrix, by Thermo Fisher Scientific (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Cellsave preservative tube, by Menarini Silicon Biosystems (1 mentions)
Facs lsr 2, by BD (1 mentions)
Edta coated tube, by Sarstedt (1 mentions)
Fc block, by BioLegend (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Wright giemsa stain, by Merck Group (1 mentions)
Tnf α elisa kit, by R&D Systems (1 mentions)
Whatman 903, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
20 protocols using edta coated vacutainer
1
Isolation and Cryopreservation of PBMCs
2
Stool and Blood Sample Collection
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Stool samples were obtained with informed consent from healthy individuals and the approval of the Research Ethics Committee Office of the National Taiwan University Hospital under project number 202006060RINB. Three aliquots of stool samples were collected at the same time point and stored in a sterile tube. The samples were transported to the laboratory and stored at − 80°C until isolation, limiting the number of freeze‒thaw cycles to a maximum of one and avoiding under room temperature for more than four hours. Whole blood was collected from donors using a 21 G needle into EDTA-coated vacutainers (BD Biosciences, San Jose, CA). Within 1 hour of collection, the subsequent purification steps for PBMCs were performed as described below.
3
Breast Cancer Liquid Biopsy Study
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All liquid biopsy collection and preparation procedures were approved by the Institutional Review Board (IRB0006379) of Ain Shams University, Cairo, Egypt. Following the Declaration of Helsinki, all participants signed an informed consent to be enrolled in this investigation. Patients included were neither pregnant nor issued with bloodborne or autoimmune disease, while healthy subjects had no oncologic history. This prospective case-control study included 77 women; 57 were diagnosed with breast cancer (34 non-IBC and 23 IBC), while 20 were healthy volunteers used as control group. This study followed two consecutive phases: procedure optimization (n = 12) and biomarker screening phase (n = 77). Peripheral blood was collected on the same day of the curative surgery in EDTA coated vacutainers (BD Bioscience, CA, USA) and centrifuged for 10 min at 1,500 g to isolate plasma. All clinicopathological data including age, tumor size, tumor grade, lymphovascular invasion, lymph node metastasis, estrogen receptor (ER) status, progesterone receptor (PR) status, and human epidermal growth receptor 2 (HER-2) were collected from the clinical and pathological records.
4
Isolation of Murine Immune Cells
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Spleen harvested from euthanized mice were homogenized and filtered through a 40-micron cell strainer. The cells were then washed with PBS, depleted of erythrocytes with ACK lysis buffer, washed twice with PBS, and resuspended in FACS buffer.
For PBMCs, whole blood was collected from mice in EDTA-coated vacutainers (BD Biosciences, San Jose, CA). The blood was mixed with PBS and centrifuged at 300 g for 10 min. The cells were then resuspended in ACK lysis buffer, washed twice with PBS, and resuspended in FACS buffer.
For PBMCs, whole blood was collected from mice in EDTA-coated vacutainers (BD Biosciences, San Jose, CA). The blood was mixed with PBS and centrifuged at 300 g for 10 min. The cells were then resuspended in ACK lysis buffer, washed twice with PBS, and resuspended in FACS buffer.
5
Hippocampus Extraction and DNA/RNA Isolation
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Right after the last memory test, rats were sacrificed by decapitation and hippocampus were dissected out, frozen on dry ice, and stored at − 70 °C for later use. Blood samples were collected retro-orbitally under isoflurane anesthesia into EDTA-coated Vacutainers (BD Diagnostics, Franklin Lakes, New Jersey) 24-h post FPI, and immediately centrifuged at 1600g for 10 min at 4 °C. The buffy coat layer containing leukocytes and platelets was carefully transferred to a new tube without disturbing the underneath leukocytes, and washed twice with 15 ml of EL Buffer (QIAGEN GmbH, Hilden, Germany) for 15 min at 4 °C. Total cellular DNA and RNA were extracted using the AllPrep DNA/RNA mini kit (QIAGEN GmbH, Hilden, Germany).
6
Fasting Blood Sampling and Storage
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Blood was drawn into EDTA-coated vacutainers (BD Biosciences, San Jose, CA, USA) via venous catheter while subjects had been fasting for the prior 12 hours. Samples were centrifuged at 4 °C for 15 min at 1700×g and the plasma stored at – 80 °C until analysis.
7
Spinal Cord Tissue Extraction and Preservation
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At the experimental endpoint, mice were euthanized via isoflurane overdose. For RNA quantification experiments, blood was collected from the left ventricle into EDTA-coated vacutainers (BD Biosciences, Mississauga, Canada) and centrifuged at 1500g for 10 min to separate plasma from the cellular pellet. Plasma was then aliquoted and snap frozen in liquid nitrogen prior to storage at −80°C. Mice were transcardially perfused with 1× PBS to clear tissues of blood, and the spinal cords were extracted, frozen on liquid nitrogen and stored at −80°C. For immunohistochemistry, mice were transcardially perfused with 1× PBS, then with ice cold 4% (w/v) paraformaldehyde in 1× PBS. Spinal cords were post-fixed in 4% paraformaldehyde with 10% (w/v) sucrose for 8–16 h at 4°C, followed by a PBS wash and overnight cryoprotection in a 30% (w/v) sucrose in 1× PBS solution. Spinal cords were then embedded in Shandon™ M-1 embedding matrix (ThermoFisher Scientific, Waltham, MA, USA), sliced into 30-μm cross-sections on a cryostat (Leica Microsystems Canada, Richmond Hill) and mounted on Superfrost Plus microscope slides (ThermoFisher Scientific, Waltham, MA, USA).
8
Blood Collection and PBMC Isolation for ccRCC
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Blood was collected from 14 patients undergoing radical nephrectomy for clear-cell renal cell carcinoma (ccRCC). Demographics and clinicopathological characteristics can be found in Table 1. All samples were collected prior to surgery (baseline) and at 3-month post-surgery with approved Mayo Clinic Institutional Review Board (IRB #16-006956) and in accordance with the World Medical Association's Declaration of Helsinki (most recent version). Whole patient blood was drawn in EDTA-coated vacutainers (BD Biosciences, San Jose, CA). Peripheral blood mononuclear cells (PBMC) were isolated from patient blood by centrifugation with Lymphoprep (StemCell Technologies, Vancouver, Canada) and SepMate devices (StemCell Technologies, Vancouver, Canada). PBMC were resuspended in Cryostor freeze medium and stored in liquid nitrogen until use. Platelet-poor plasma was obtained from centrifugation of whole blood at 2,500 × g for 15 minutes at room temperature. Plasma was aliquoted in cryovials and stored at -80°C until use.
9
Measuring CGRP Levels in Plasma
10
Neutrophil Isolation from Whole Blood
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Healthy blood donors were consented under the Massachusetts General Hospital Institutional Review Board-approved protocol (2019P002840). Whole blood was collected in EDTA-coated vacutainers (Beckton Dickinson, Franklin Lakes, NJ, USA) and subsequently centrifuged at 1500xg for 15 min. Buffy coat was collected, and neutrophil isolation was performed using the negative selection EasySep Direct Human Neutrophil Isolation Kit, according to the manufacturer’s instructions (STEMCELL Technologies, Seattle, WA, USA). Wright-Giemsa staining was performed after the isolation process to confirm neutrophil purity from the isolation kit. Flow cytometry was also used to verify a high neutrophil purity from the isolation procedure (≥94% neutrophil purity). Cell concentration and viability were measured by staining the cells with a 1:10 dilution of acridine orange/propidium iodide followed by automatic cell counting using the LUNA fl Dual Fluorescence Cell Counter (Logos Biosystems, Annandale, VA, USA) (≥99% live). Neutrophils were resuspended in cRPMI at a concentration of 2 × 106 cells/mL.
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