Venous blood samples were collected from 24 healthy volunteers (control) and 120 CRF patients who represented 5 different stages (n=24 in each stage) according to their glomerular filtration rates (GFR): 1 (GFR ≥ 90 mL/min/1.73 m2); 2 (60 < GFR< 89 mL/min/1.73 m2); 3 (30 < GFR< 59 mL/min/1.73 m2); 4 (15 < GFR< 29 mL/min/1.73 m2); and 5 (GFR < 15 mL/min/1.73 m2) 7 (link). All patients were diagnosed with CRF and received therapy in the Department of Nephrology, Jiangxi Provincial People's Hospital, during 2012-2016, and their basic characteristics are summarized in Supplementary Table 1 . Blood samples were kept in K2EDTA-coated tubes (BD, Franklin Lakes, NJ, USA, #367835) until use. All participants signed a consent form reviewed and approved by the ethical board of Jiangxi Provincial People's Hospital in China. The serum concentration of TGF-β was determined by an ELISA kit (ThermoFisher Scientific, #BMS249-4).
K2edta coated tubes
Manufactured by BD
Sourced in United States
K2EDTA-coated tubes are laboratory consumables used for the collection and stabilization of blood samples. The tubes are coated with the anticoagulant K2EDTA, which prevents the blood from clotting, allowing for accurate testing and analysis of the sample.
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Lab products found in correlation
C57bl 6 mice, by Jackson ImmunoResearch (2 mentions)
Bms249 4, by Thermo Fisher Scientific (1 mentions)
Legendplex mouse anti virus response panel, by BioLegend (1 mentions)
Prism v10, by GraphPad (1 mentions)
Legendplex mouse cytokine panel 2, by BioLegend (1 mentions)
Pharm lyse, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Microhematocrit capillary tube, by Thermo Fisher Scientific (1 mentions)
Cd45 pe cy7, by BioLegend (1 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa facs machine, by BD (1 mentions)
Cd11b af700, by Thermo Fisher Scientific (1 mentions)
Influx sorter, by BD (1 mentions)
Fc receptor binding inhibitor, by Thermo Fisher Scientific (1 mentions)
F4 80 apc, by BioLegend (1 mentions)
Dnase, by Roche (1 mentions)
Collagenase a, by Roche (1 mentions)
Tnf α, by Eve Technologies (1 mentions)
Mip 2, by Eve Technologies (1 mentions)
Rantes, by Eve Technologies (1 mentions)
14 protocols using k2edta coated tubes
1
Serum TGF-β Levels in CRF Patients
2
PBMC Isolation from Peripheral Blood
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Peripheral venous blood was taken into the K2 EDTA coated tubes (BD, Franklin Lakes, NJ). Within one hour of blood draw, PBMCs were isolated as described by Huang et al. [15 (link)]. PBMCs samples were stored at −80 °C until the total RNA extraction.
3
Cytokine Profiling in Mouse Models
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Blood was collected by either cheek bleed or cardiac puncture in K2EDTA-coated tubes (BD Biosciences). Tubes were centrifuged at 2000 × g for 15 min at 4 °C, and the plasma was collected for analysis. Cytokine levels were evaluated using either the LEGENDplex™ Mouse Anti-Virus Response Panel (BioLegend) or the LEGENDplex™ Mouse Cytokine Panel 2 (BioLegend), both with V-bottom plates, according to manufacturer’s instructions, and data were collected using flow cytometry. Cytokine concentrations were interpolated from standard curves using an asymmetric sigmoidal 5-paramater logistic curve fits (GraphPad Prism V10). Bar plots comparing groups and heat maps of averaged values for groups were generated to analyze results.
4
Time-course Analysis of AMI Biomarkers
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All peripheral venous blood samples were obtained from the AMI patients upon admission to Union hospital. The initial blood sample collection time (T0) was 9.24 ± 2.81 h after the onset of AMI symptoms. Subsequent blood samples were obtained at 4 h ± 30 min (4 h), 12 h ± 30 min (12 h), 24 h ± 30 min (24 h), 48 h ± 30 min (48 h), and 72 h ± 30 min (72 h) after T0. Additionally, plasma was collected pre- and post-PCI procedures to investigate whether heparin had an impact on the expression of selected miRNAs in plasma.
All samples (4-8 mL) were collected into K2-EDTA-coated tubes (BD, NJ, USA). The samples were stored for up to 24 h at 4°C and prepared according to the following 2-step centrifugation protocol: 1,500 x g for 15 min at 4°C, then 14,000 x g at 4°C for another 15 min. After centrifugation, the supernatant (plasma) was aliquoted into RNase/DNase-free tubes and stored at −80°C for subsequent experiments.
All samples (4-8 mL) were collected into K2-EDTA-coated tubes (BD, NJ, USA). The samples were stored for up to 24 h at 4°C and prepared according to the following 2-step centrifugation protocol: 1,500 x g for 15 min at 4°C, then 14,000 x g at 4°C for another 15 min. After centrifugation, the supernatant (plasma) was aliquoted into RNase/DNase-free tubes and stored at −80°C for subsequent experiments.
5
Plasma Collection for Transgenic Mouse Models
6
Isolation and Immunophenotyping of Murine Leukocytes
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Whole blood was collected into K2EDTA coated tubes (BD, cat# 365973) from mice by tail nick 2 days after the last intraperitoneal injection of depleting or control antibodies. The Buffy coat was isolated by spinning blood at 800×g at room temperature for 10 min and removing the small layer of the buffy coat. Any carryover red blood cells were lysed using Pharmlyse (BD, 5 min) and quenching with FACS buffer (DPBS supplemented with 2% FCS and 2% HEPES). Cells were washed and stained with BV711 conjugated anti-mouse CD8a (clone53-6.7, Biolegend, 1:200, cat# 563046), PE-conjugated anti-mouse CD4 (clone RM4-5, Biolegend, 1:200, cat# 116006), and APC conjugated anti-mouse NKp46 (clone 29A1.4, Biolegend, 1:200, cat# 137607). Cells were washed three times before staining with DAPI. Data were collected using the BD LSR Fortessa (FACSDiva 8.0.1).
7
Isolation and Analysis of Hybrid Circulating Tumor Cells
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Tumors were diced and digested for 30 min at 37°C in DMEM + Collagenase A (2 mg/ml; Roche) + DNase (Roche) under stirring conditions. Digested tumor cells were filtered through a 40-μm filter and washed with PBS. For FACS isolation, hybrid and unfused cells were isolated by direct fluorescence on a Becton Dickinson InFlux sorter. For flow cytometric analysis, blood was collected retro-orbitally using heparinized microhematocrit capillary tubes (Fisher) into K2EDTA-coated tubes (BD Biosciences). RBC lysis was performed as described above. Cells were washed and resuspended in FACS buffer (PBS, 1.0 mM EDTA, and 5% FBS). Cells were incubated in PBS containing LIVE/DEAD Fixable Aqua (1:500; Invitrogen) with Fc Receptor Binding Inhibitor (1:200; eBioscience). Cells were then incubated in FACS buffer for 30 min with CD45-PeCy7 (1:8000; BioLegend), CSF1R-BV711 (1:200; BioLegend), F4/80-APC (1:400; BioLegend), and CD11b-AF700 (1:200; eBioscience). A BD LSRFortessa FACS machine was used for analyses. A statistical significance of P < 2.2 × 10−6 by unpaired t test was determined for CD45+ hybrid CTCs relative to CD45− hybrid, CD45+ unfused, and CD45− unfused CTCs. Technical duplicates of n = 5 or 6 mice were analyzed.
8
Plasma Cytokine Profiling in Mice
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Blood was collected from the submandibular vein of all experimental mice into K2EDTA-coated tubes (BD Biosciences, 365974) and immediately placed on ice. The blood was centrifuged for 15 minutes at 900g and 4°C, and plasma was collected. Aliquots of plasma were diluted twofold in PBS and stored at –80 °C. Blood plasma cytokines were analysed through a commercially available multiplex panel including Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, CXCL1, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1alpha, MIP-1beta, MIP-2, RANTES, TNF-α, and VEGF (Eve Technologies, Alberta, Canada, MD-31).
9
Plasma Collection from Genetically Altered Mice
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12-week-old male C57BL/6 mice were purchased from Jackson Laboratories. All animal work was performed in accordance with the University of Southern California Institutional Animal Care and Use Committee and Animal Research Committee (ARC) protocols of the University of California, Los Angeles and according to NIH guidelines. Mice were intraperitoneally injected for 20 days with recombinant human IGF-I (rhIGF-I) at 500 μg kg−1 day−1 (BID) and recombinant human (rhGH) at 2 mg kg−1 day−1. Following 20 days of rhGH and rhIGF-I treatment, blood collected in K2-EDTA coated tubes (BD, Franklin Lakes, NJ, USA) by cardiac puncture at the time of euthanasia were immediately centrifuged for 10 min at 1500 g using a refrigerated centrifuge to obtain plasma. Plasma from 16-week-old male LID mice, 16-week-old female PEPCK-bGH mice, 24-week-old male Ames mice, 11-week-old male IGFBP-3 KO (BP3KO) mice, and their respective age- and sex-matched matched controls were collected using the same procedures. All genetically altered mice were of the C57BL/6 background.
10
Cytokine Profiling in Murine Blood and Peritoneal Fluid
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Whole blood obtained control and experimental mice was collected in K2EDTA-coated tubes (365974, BD Sciences, ON Canada). The blood was centrifuged for 15 min at 900 g and 4°C, plasma was then isolated, aliquoted and stored at −80°C. Using a mouse multiplex cytokine and chemokine array (MD31; Eve Technologies, Calgary, AB, Canada), cytokines were analyzed using the same mouse panel described above. Peritoneal lavage fluid (PF) was collected at the time of sacrifice via peritoneal lavage with 5 mL of ice-cold PBS and immediately put on ice. PF was centrifuged for 5 min at 400 g and 4°C, then PF was aliquoted and stored at −80°C. Cytokines were assessed with the same mouse multiplex cytokine and chemokine array as described above.
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