Total blood counts, leucocyte, and monocyte counts were measured with a Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Undiluted blood (50 µL) was incubated for 15 min in the dark at room temperature with the following antibodies: CD3-APC-750 (mouse, dilution: 1:25; Beckman Coulter, Woerden, The Netherlands), CD56-APC (mouse, 1:25; Beckman Coulter), CD16-FITC (mouse, 1:25; eBioscience Thermo Fisher Scientific, Breda, The Netherlands), CD14-PC7 (mouse, 1:25; eBioscience Thermo Fisher Scientific), CCR2-BV421 (mouse, 1:50 and 1:25; BD Biosciences), CD36-APC-700 (mouse, 1:10; Beckman Coulter), CD41-PC5.5 (mouse, 1:50; Biolegend, San Diego, CA, USA), CD11b-BV785 (mouse, 1:50 and 1:25; Biolegend). Followed by the addition of 1 mL 10× diluted lysis buffer (BD Pharm Lyse; BD Biosciences), samples were mixed and incubated for 10 min in the dark at room temperature. Samples were then measured on a Beckman Coulter FC500 flow cytometer, each sample was measured both stained and unstained. Flow cytometry data were analysed using Kaluza software (Beckman Coulter); for gating strategy, see Supplementary material online , Figure S1 . Besides determining the different monocyte subsets (classical, intermediate, or non-classical). Atherogenic markers CCR2, CD36, CD41, and CD11b were determined per monocyte subset.
Cd3 apc 750
Manufactured by Beckman Coulter
Sourced in Netherlands
The CD3-APC-750 is a fluorescently labeled antibody that binds to the CD3 antigen on T cells. It is designed for use in flow cytometry applications to identify and quantify T cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 apc, by Beckman Coulter (2 mentions)
Ccr2 bv421, by BD (2 mentions)
Cd36 apc 700, by Beckman Coulter (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Pharm lyse, by BD (2 mentions)
Cd11b bv785, by BioLegend (1 mentions)
Xn 450 hematology analyzer, by Sysmex (1 mentions)
Cd16 fitc, by BD (1 mentions)
Hla dr pe, by Beckman Coulter (1 mentions)
Cd14 pc7, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Cd45 ko, by Beckman Coulter (1 mentions)
Xn 450, by Sysmex (1 mentions)
Xn 9000, by Sysmex (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Cd4 apc, by Beckman Coulter (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
3 protocols using cd3 apc 750
1
Comprehensive Monocyte Phenotyping by Flow Cytometry
2
Immune cell profiling using flow cytometry
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Immune cell subset numbers were measured on a Sysmex XN-450 and Sysmex XN-9000 (Sysmex). FACS analysis was performed in one of the two participating study sites, because this method is sensitive to confounders when performed at different sites. A total of 50 μl of whole undiluted blood was incubated for a duration of 15 min in the dark at room temperature with the following antibodies: CD16-FITC (dilution 1:20), CD14-PC7 (1:20), CCR2-BV421 (1:20) (BD Biosciences, Vianen, the Netherlands); CD41-PC5.5 (1:20), CD11b-BV785 (1:20) (ITK Diagnostics BV, Uithoorn, the Netherlands); HLA-DR-PE (1:10), CD56-APC (1:10), CD3-APC-750 (1:10), CD45-KO (1:10), CD36-APC-700(1:10) (Beckman Coulter, Woerden, the Netherlands). Subsequently, 1 ml of lysis buffer (BD Pharm Lyse, BD Biosciences) was added, samples were mixed, incubated for another 10 min and then measured on a flow cytometer (Beckman Coulter FC500). To determine the position of analysis gates, single staining and fluorescence-minus-one control stains were used (Additional file 1 : Fig. S1). To analyse the FACS data, Kaluza software (Beckman Coulter, Woerden, the Netherlands) was used.
3
Polyclonal Expansion of Human Regulatory T Cells
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Freshly isolated Tregs were polyclonally expanded as described previously (13 (link)). Briefly, Tregs were cultured in X-VIVO15 medium (Lonza, Basel, Switzerland) containing 10% serum, 500 U/mL human recombinant IL-2 (Chiron Behring, Marburg, Germany) and 100 nM rapamycin (Sigma–Aldrich, St. Louis, MO) and stimulated with CD3/CD28 Treg expansion beads (Miltenyi Biotech). Cells were restimulated every 3-4 days. After 3-weeks expansion time tTreg product purity was assessed by specific staining of CD3-APC750 (Clone UCHT1), CD4-APC (Clone 13B8.2), CD8-A700 (Clone B9.11), CD25-PeCy7 (Clone B1.49.9) from Beckman Coulter, Krefeld Germany) and FoxP3-A488 (Clone 259D/C7, Beckton Dickinson, Heidelberg, Germany). Mean Treg purity after isolation was 80% for CD3+CD4+ and 60% for CD3+CD4+CD25high cells. After isolation Tregs were expanded in cell culture medium supplemented with IL-2 and rapamycin in order to promote Treg expansion and suppress expansion of non-Treg cells. The final Treg products consisted of ~90% CD3+CD4+ T-cells with a frequency of 94% CD25highFoxP3+ Tregs.
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