Non-lethal Plasmodium yoelii 17XNL (Py17XNL) and Plasmodium chaubadi chaubadi AS (PcAS) parasites were used following one in vivo passage in wild type C57BL/6J mice. Using parasitized red blood cells (pRBC) that were obtained from frozen/thawed stabilates, mice were infected i.v. with either 104 pRBCs (Py17XNL) or 105 pRBCs (PcAS). Blood parasitemia was measured in Diff-Quick (Lab Aids, Narrabeen, NSW, Australia) or giemsa-stained thin blood smears obtained from tail bleeds. Alternatively, a modified protocol of a previously established flow cytometric method was employed to measure parasitemia more rapidly [66 (link)]. Briefly, a single drop of blood, from a tail bleed or cardiac puncture, was diluted and mixed in 250μl RPMI containing 5U/ml heparin sulphate. Diluted blood was simultaneously stained with Syto84 (5μM; Life Technologies) to detect RNA/DNA, and Hoechst33342 (10μg/ml; Sigma) to detect DNA, for 30 minutes, in the dark at room temperature. Staining was quenched with 10x volume of ice cold RPMI, and samples were immediately analysed by flow cytometry, using a BD FACSCantoII analyser (BD Biosciences) and FlowJo software (Treestar, CA, USA). pRBC were readily detected as being Hoechst33342+ Syto84+, with white blood cells excluded on the basis of size, granularity and much higher Hoechst33342/Syto84 staining compared to pRBC.
Facscanto 2 analyser
Manufactured by BD
Sourced in United States
The BD FACSCanto II is a flow cytometry analyser designed for multicolor flow cytometry applications. It features a blue (488 nm) and red (633 nm) solid-state laser configuration and can detect up to 8 fluorescent parameters simultaneously. The instrument is capable of analysing a wide range of sample types, including cells, microparticles, and bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Syto84, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Violet dead cell stain, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Cellrox orange reagent, by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Cd44 apc, by Thermo Fisher Scientific (1 mentions)
Cd62l fitc, by BD (1 mentions)
Cd8a efluor 450, by Thermo Fisher Scientific (1 mentions)
Cd3e pe, by BD (1 mentions)
Rbc lysis buffer, by BioLegend (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
Anti mouse igg1 pe, by BD (1 mentions)
Fitc conjugatedanti mouse igg2a, by BD (1 mentions)
Apc conjugated cd90, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Mouse igg1, by BD (1 mentions)
9 protocols using facscanto 2 analyser
1
Rapid Malaria Parasitemia Quantification
2
Cytokine Profiling of Antigen-Stimulated PBMCs
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Purified PBMCs (2 x 106 cells/ml) were cultured in the presence of 1 μg/ml of fusion protein and 1 μg/ml MAP3694c protein for 6 hours followed by 16 hours in the presence of Brefeldin A (10 μg/ml; Sigma) and Monensin (2 μM/ml; Sigma). Control cultures containing either medium alone or mitogen (1 μg/ml of SEB) were run in parallel. After washing cultured PBMCs with PBS (supplemented with EDTA and sodium azide) the cells were stained. Anti-CD4 antibody (clone IL-A11; Veterinary Medical Research & Development, VMRD, Pullman, WA, USA) was used for surface stain in combination with violet dead cell stain (Invitrogen) before the cells were intracellularly stained with antibodies specific for IFN-γ (PE-conjugated, clone CC302; Bio-Rad), TNF-α (AF488-conjugated, clone CC327; Bio-Rad), and IL-2 (DyLight649-conjugated, Clone 86; a kind gift from Adam Whelan, APHA, UK) [16 (link)]. Using a BD FACSCanto II analyser equipped with 405, 488 and 633 nm lasers (BD Bioscience, USA) flow data were acquired and analyzed with BD FACSDiva software vs. 6.1.2 (data in S4 Table ).
3
Flow Cytometry Gating and Analysis
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Data were acquired and analysed on a BD FACSCanto II analyser using FASCDIVA software version 6.1.3. Fluorescence Minus One (FMO) and unstimulated controls were used for creating gating templates and all gating and analyses were done in FACSDiva. The flow cytometer was calibrated on each day that samples were processed using Cytometer Setup & Tracking beads kit (BD Biosciences). Single colour compensation controls (CompBead Anti-Mouse Ig, negative control particles set) (BD Biosciences) were applied to each experiment and application settings were used for the study. Flow cytometry gating was reviewed and independently agreed upon by three laboratory scientists for consistency and to minimize bias.
4
Quantifying Intracellular ROS in PBMCs
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Blood samples have been collected in fasting conditions in lithium heparin tubes. Blood was immediately centrifuged, and plasma samples were stored at −80oC for a maximum period of 6 months. We processed 4 cc of plasma in order to analyse:
CellROX® Orange Reagent (Life Technologies, Carlsbad, California, USA) was used for measuring intracellular reactive oxygen species (ROS) production. Peripheral blood mononuclear cells (PBMCs) were isolated by stratifying heparinized whole blood on Ficoll-Hypaque (GE Healthcare, Chicago, Illinois, USA). Freshly isolated PBMCs were incubated with 5 µM CellROX® Orange Reagent for 30 min in the dark at 37°C, washed three times and re-suspended in phosphate-buffered saline solution (PBS)). The fluorescence was quantified using FACScanto II analyser (Becton-Dickinson, San Diego, CA, USA) and Flow-Jo software (Tree Star Inc., Ashland, OR, USA); intracellular ROS production (CellROX) was measured as percent positive cells (%) and mean fluorescence intensity (MFI).
CellROX® Orange Reagent (Life Technologies, Carlsbad, California, USA) was used for measuring intracellular reactive oxygen species (ROS) production. Peripheral blood mononuclear cells (PBMCs) were isolated by stratifying heparinized whole blood on Ficoll-Hypaque (GE Healthcare, Chicago, Illinois, USA). Freshly isolated PBMCs were incubated with 5 µM CellROX® Orange Reagent for 30 min in the dark at 37°C, washed three times and re-suspended in phosphate-buffered saline solution (PBS)). The fluorescence was quantified using FACScanto II analyser (Becton-Dickinson, San Diego, CA, USA) and Flow-Jo software (Tree Star Inc., Ashland, OR, USA); intracellular ROS production (CellROX) was measured as percent positive cells (%) and mean fluorescence intensity (MFI).
5
FACS-Based Cell Analysis Protocol
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FACS analyses were performed by standard methods. Briefly, 1 x 10 6 test cells per group resuspended in FACS buffer (BSA (1 % w/v); EDTA (2 mM). The cells were blocked in FACS buffer containing human serum (10 % v/v) for 15 min prior to incubation for 45 min with relevant antibodies (see table 1). Cells were then analysed using a FACSCanto II analyser (Becton Dickinson, USA). Samples were acquired using CellQuest pro software (Becton Dickinson, USA) and analysed using WinMDI 2.8 software. Cell viability was assessed by propidium iodide uptake (20 μg/ml for 10 min) via FACS analysis and was found to be > 90% in all cases.
6
Propidium Iodide Viability Assay
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The uptake of the fluorescent restriction dye Propidium iodide (PI) was used to measure the viability of cells under the experimental procedures described above. After 24h post-culture monocytes were incubated in PBS containing PI (10μg/ml) for 10 min. The number of nonviable cells (PI +) was assessed uaing FACSCanto II analyser (Becton Dickinson, USA). Samples were acquired using BD FACSDiva™ (BD Biosciences, USA) and analysed using CyFlogic 2.8 software, licensed to Nottingham University. Monocytes which had been immersed in ice cold (-20 0C) methanol for 30 min were used as a positive control and monocytes cultured in media only for 24h were used as a negative control. All experiments were performed in triplicate on 3 separate occasions.
7
Quantification of T cell subsets
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Blood cells from 300 μl blood were incubated in RBC-Lysis buffer (Biolegend) to lyse the red blood cells. Remaining cells were washed and incubated with a cocktail of fluorochrome-conjugated antibodies (Cd4-PE-Cy7 (#552775) and Cd62L-FITC (#561917) from BD Pharmingen; Cd3e-PE (#12–0031), Cd8a-eFluor 450 (#48–0081) and Cd44-APC (#17–0441) from eBioscience.), incubated with propidium iodide for the detection of dead cells and analysed using the FACSCanto II analyser (BD Biosciences). The following T cell subsets were quantified: Cd3+, Cd8+, Cd44high cytotoxic memory T cells; Cd3+, Cd8+, Cd44low, Cd62Lhigh cytotoxic naïve T cells, Cd3+, Cd4+, Cd44high helper memory T cells and Cd3+, Cd4+, Cd44low, Cd62Lhigh helper naïve T cells.
8
Immunophenotyping of CD49f- and CD271+CD49f- Cells
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FACS sorted CD49f- cells or cultured CD271+CD49f- cells were stained with primary antibodies PE-conjugated CD271, PE-conjugated PDGFRB (1.25 μg/ml, mouse IgG1; R&D Systems), PE-conjugated SUSD2, (W5C5 clone, 1:20, mouse IgG1; Biolegend), APC-conjugated CD90 (25 μg/ml, mouse IgG1κ, BD Pharmingen), CD146 (1:2, supernatant, clone CC9, mouse IgG2a; donated by Prof David Haylock, CSIRO, Clayton, Victoria, Australia), CD73 (10 μg/ml, mouse IgG1κ; BD Pharmingen) or CD105 (10 μg/ml, mouse IgG1ƙ; BD Pharmingen). The CD146 samples were incubated with secondary antibody fluorescein isothiocyanate (FITC) conjugated anti-mouse IgG2a (5 μg/ml, clone R19-15; BD Pharmingen). CD73 and CD105 samples were incubated with secondary antibody PE-anti-mouse IgG1 (2 μg/ml, clone A85-1, BD Pharmingen). Isotype matched controls or unlabelled controls were included for each antibody and used to set the electronic gates on the flow cytometer. Cells were then incubated with Sytox Blue and analysed by FACS Canto II analyser (BD Biosciences). FACS data were analysed by FACSDiva software (BD Biosciences).
9
Profiling Innate Immune Responses in Whole Blood
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Fluorochrome-conjugated monoclonal antibodies (anti-human CD16, CD11b, IL-8, TLR-2, TLR-4, and TLR-9; BD UK) were used for staining individual patient polymorphonuclear leucocytes from whole blood and analysed by flow cytometry using a FACS Canto II analyser and FACS Diva 6.1.2 software (BD, San Jose, CA). 50,000 granulocytes were gated on forward and side-scatter characteristics and stained with anti-CD16-phycoerythrin-IgG1j. Fluorochrome mean fluorescence intensity was calculated to detect the receptor binding response and to measure antigen-antibody binding. Neutrophil oxidative burst was quantified using Glycotope Biotechnology Phagoburst TM (BD Biosciences) kits measuring the percentage of phagocytic cells producing ROS at rest. 16 The formation of ROS was detected using the oxidation of dihydrorhodamine-123 to rhodamine-123. Neutrophil phagocytic activity was assessed by neutrophil phagocytosis of opsonized Escherichia coli. 16 Plasma cytokine profiling Plasma cytokines were measured using the Meso Scale Discovery (MSD) platform. Samples were run in duplicate on U-PLEX Proinflam Combo 1 (hu) plates, measuring interferon-c (IFN-c), interleukin (IL)1-b, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12 p70, IL-13, and TNF-a.
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