Blood samples were collected from all rats and divided to fill 2 0.5 ml K3EDTA-containing tubes (Becton–Dickinson, Franklin Lakes, NJ). Samples were analyzed on an impedance hematology analyzer. The following variables were assessed: red blood cell count, hemoglobin concentration, mean corpuscular volume, total white blood cell count, percentage of neutrophils, lymphocytes, monocytes, and eosinophils. For blood gases, the samples were analyzed within 5 min as recommended by i-Stat1 technical bulletin using CG8 cartridges. All variables were determined using the VetScan i-Stat1 handheld analyzer (Abaxis, Union City, CA). The pH (hydrogen potential), pCO2, pO2, bases excess, HCO3, Na+, and K+ were carried out in all blood samples.
K3edta containing tubes
Manufactured by BD
Sourced in United States
K3EDTA-containing tubes are laboratory equipment used for the collection and preservation of blood samples. The primary function of these tubes is to contain the anticoagulant K3EDTA, which prevents blood from clotting during the sample collection process.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll histopaque, by Merck Group (4 mentions)
Midimacs system, by Miltenyi Biotec (4 mentions)
Facsdiva software, by BD (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Trypan blue exclusion, by Merck Group (2 mentions)
Vetscan i stat 1 handheld analyzer, by Abaxis (1 mentions)
Dextran sedimentation, by Merck Group (1 mentions)
6 protocols using k3edta containing tubes
1
Hematological and Blood Gas Analysis
2
Monocyte Isolation and Treatment Protocol
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This study was conducted according to Good Clinical Practice Guidelines and in line with the principles outlined in the Helsinki Declaration of the World Medical Association. Informed consent for the study was obtained from healthy male blood donors (age <35 years) at the University Hospital Virgen del Rocio, Seville. Participants declared that they were non-smokers and were not taking any medication. Peripheral blood samples were drawn from a large antecubital vein and collected into K3EDTA-containing tubes (Becton Dickinson, NJ, USA).
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples by centrifugation over a Ficoll-Histopaque (Sigma-Aldrich, Madrid, Spain) gradient. Monocytes were isolated from PBMCs using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain) according to the manufacturer's instructions. The purity for CD14 monocyte isolations was routinely >90% by flow cytometry (FACScanto II flow cytometer and FACSDiva software, BD). Following isolation, monocytes were suspended in a RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin and 10% heat-inactivated foetal bovine serum. 14 For treatments, 5 × 10 5 of purified monocytes, after in vitro stimulation with or without LPS (100 ng/mL), were exposed to GSOUF at 10-100 mM for 24 h.
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples by centrifugation over a Ficoll-Histopaque (Sigma-Aldrich, Madrid, Spain) gradient. Monocytes were isolated from PBMCs using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain) according to the manufacturer's instructions. The purity for CD14 monocyte isolations was routinely >90% by flow cytometry (FACScanto II flow cytometer and FACSDiva software, BD). Following isolation, monocytes were suspended in a RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin and 10% heat-inactivated foetal bovine serum. 14 For treatments, 5 × 10 5 of purified monocytes, after in vitro stimulation with or without LPS (100 ng/mL), were exposed to GSOUF at 10-100 mM for 24 h.
3
Isolation of Monocytes from Peripheral Blood
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The same six volunteers who took part as donors of postprandial TRLs participated as donors of monocytes. After an overnight fasting period of 12 h, peripheral blood samples were drawn from a large antecubital vein and collected into K3EDTA-containing tubes (BD). Peripheral blood mononuclear cells (MNCs) were isolated by centrifugation over a Ficoll–Histopaque (Sigma, Madrid, Spain) gradient [19 (link)]. Monocytes were isolated from peripheral blood MNCs using anti-CD14 microbeads and LS columns on a midiMACS system (MiltenyiBiotec, Madrid, Spain). Monocyte (CD14+) purity was routinely >90% by flow cytometry analysis (FACSCanto II flow cytometer and FACSDiva software, BD) and cell viability >95% by trypan blue exclusion (Sigma). The monocytes were seeded in 24-well culture plates at a density of 1 × 106 cells/mL and cultured in ultra-low attachment flasks in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum (complete culture medium).
4
Isolation of Monocytes from Peripheral Blood
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The same six volunteers who took part as donors of postprandial TRLs participated as donors of monocytes. After an overnight fasting period of 12 h, peripheral blood samples were drawn from a large antecubital vein and collected into K 3 EDTA-containing tubes (BD). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Histopaque (Sigma) gradient. Monocytes were isolated from PBMCs using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec). Monocytes were tested for purity by CD14 fluorescein isothiocyanate (FITC) labelling and fluorescence-activated cell sorter (FACS) analysis. The detailed method can be found in the Supporting Information.
5
Macrophage Differentiation in Postprandial States
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The same six volunteers who took part as donors of postprandial TRLs or fasting leukocytes participated in this part of the study. After an overnight fasting period of 12 h, all of them were given, over three different occasions, an oral fat emulsion (meal rich in SFAs, meal rich in MUFAs or meal rich in MUFAs plus omega-3 LCPUFAs) as indicated above and a single dose of 2 g immediate-release niacin (Twinlab, UT, USA). The participants also consumed the same test meal without fat (including niacin), as a control meal. Peripheral blood samples were drawn from a large antecubital vein at fasting and at the postprandial hypertriglyceridemic peak, i.e. 2-3 h, and collected into K 3 EDTA-containing tubes (BD). Monocytes were then isolated and differentiated into naïve GM-M0 and M-M0 macrophages as described above.
6
Isolation and Culture of Immune Cells
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The same six volunteers who took part as donors of postprandial TRLs participated as donors of leukocytes. After an overnight fasting period of 12 h, peripheral blood samples were drawn from a large antecubital vein and collected into K3EDTA-containing tubes (BD). Peripheral blood mononuclear cells (MNCs) were isolated by centrifugation over a Ficoll-Histopaque (Sigma, Madrid, Spain) gradient. Monocytes were isolated from peripheral blood MNCs using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain). Neutrophils were isolated by dextran sedimentation (2% dextran/0.9% NaCl) (Sigma) from the fraction of peripheral blood polymorphonuclear cells (PMNCs). Residual erythrocytes were removed using hypotonic lysis with 0.2% and 1.6% saline solutions. Monocyte (CD14 + ) and neutrophil (CD16 + ) purity was routinely >90% by flow cytometry analysis (FACScanto II flow cytometer and FACSDiva software, BD) and cell viability >95% by trypan blue exclusion (Sigma). The monocytes and neutrophils were seeded at a density of 510 5 cells/mL and 310 6 cells/mL, respectively, and cultured in ultra low attachment flasks in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, and 10% heat-inactivated fetal bovine serum (complete culture medium). PGD2 was determined in the medium of monocytes and neutrophils after 30 min.
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