Blood samples for hematology (in EDTA tubes, Microvette CB300, Sarstedt, Nürnbrecht, Germany) were collected from the left atrium of the heart under isoflurane anesthesia, prior to necropsy. Leucocytes and erythrocytes were isolated by centrifugation and stained with 1:50 dilutions of MS CD45 HRZN V500 mAb (#561487; BD Diagnostics, Stockholm, Sweden), MS F4/80 PE T45-2342 (#565410), MS CD4 PERCP mAb (#561090), MS CD19 APC mAb (#561738), CD8 APC-Cy7 mAb (#561967), NK1.1 FITC mAb (#553164), and CD3e conjugated to BD Horizon V450 (#560804). Erythrocytes were lysed with BD FACS lysis buffer and analyzed using flow cytometry (FACS Fortessa, BD Bioscience, Stockholm, Sweden) with appropriate filter settings, gating on live cells.
Horizon v450
Manufactured by BD
Sourced in United States
The BD Horizon V450 is a multi-color flow cytometry instrument designed for research applications. It is capable of detecting up to 18 parameters simultaneously. The core function of the BD Horizon V450 is to analyze and sort cells based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Horizon v500, by BD (3 mentions)
Facs fortessa, by BD (1 mentions)
Facs lysis buffer, by BD (1 mentions)
Microvette cb 300, by Sarstedt (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Alexa fluor 700, by BD (1 mentions)
Lsrfortessa machine, by BD (1 mentions)
Transcription buffer set, by BD (1 mentions)
Eflour780, by Thermo Fisher Scientific (1 mentions)
Diva software, by BD (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BD (1 mentions)
Anti foxp3, by Thermo Fisher Scientific (1 mentions)
Anti il 10, by BioLegend (1 mentions)
Anti cd62l, by Thermo Fisher Scientific (1 mentions)
Anti cd11c, by BD (1 mentions)
Anti cd8, by BD (1 mentions)
Anti cd4, by BD (1 mentions)
14 protocols using horizon v450
1
Comprehensive Immune Cell Profiling from Murine Cardiac Samples
2
Detecting IL-4Rα Expression in Immune Cells
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IL-4Rα surface expression was detected on live cells isolated from the peritoneum or lamina propria by phycoerythrin (PE) anti-CD124 (IL-4Rα, M-1). Cell subpopulations were identified with Alexa Fluor 700, BD Horizon V450, APC, or PE-Cy7 for F4/80, Ly6G, CD11c, and CD11b (BD Pharmingen). Stained cells were then acquired on a LSRII flow cytometer (BD Bioscience), and data were analyzed using FlowJo software (TreeStar Inc.).
3
Phenotyping PBMC from CF, PAH, COPD Patients
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PBMC from 7 CF, 8 PAH and 6 COPD patients and 6 HC were rapidly thawed by placing cryovials at 37°C. Cells were washed, resuspended in supplemented RPMI 1640. 3.106 cells were stained with CD3-PE-Cy7, CD4-PercP-Cy 5.5, CD127-PE, BD Horizon V450 (BD, Biosciences). Results were generated by flow cytometry (LSR II BD Biosciences and FlowJo software).
4
Multiparametric Flow Cytometry Analysis
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Il-4Rα surface expression was detected on lymph node cells by phycoerythrin (PE) anti-CD124 (IL-4Rα, M-1). Cell subpopulations were identified with Alexa Fluor 700, BD Horizon V500, BD Horizon V450, PerCP-Cy5.5, APC, APC-Cy7, Fluoroscein isothiocyanate, PE, PE-Cy7 or biotinylated monoclonal antibodies against CD3, CD4, CD19, Lineage, Gata-3, IL-4, IL-13, IFN-γ, IL-10, SiglecF, T1/ST2, ICOS. Biotin-labeled antibodies were detected by Allophycocyanin or Percpcy5.5. For staining, cells (1x 106) were labeled and washed in PBS, 3%FCS and 0.1% NaN3. Between each step of staining, cells were washed extensively. For intracellular cytokine staining, cells were restimulated with a cocktail of PMA/Ionomycin/Monensin for 4–12 h at 37°C then fixed in 2% PFA, permeabilized and cytokine production was analyzed as previously described [28 (link)]. For intranuclear staining, a commercially available transcription buffer set (BD Bioscience) was used as per the manufacturer’s instructions. All antibodies were from BD Pharmingen (San Diego, CA) except where noted otherwise. Stained cells were then acquired on a LSR Fortessa machine (BD Immunocytometry system, San Jose, CA, USA) and data were analyzed using Flowjo software (Treestar, Ashland, OR, Usa).
5
Comprehensive Immune Cell Profiling
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Anti-CD4, anti-CD8, anti-CD11c, anti-CD11b, anti-MHC-II, anti-CD80, anti-CD86, anti-CD335, anti-CD3, anti-IFN-γ (all BD Biosciences, Heidelberg, Germany), anti-CD62L, anti-TNF-α, anti-Foxp3 (all eBioscience, Frankfurt, Germany), anti-CD160, anti-CD138, and anti-IL-10 (Biolegend, London, UK) were used as fluorescein isothiocyanate, pacific blue, phycoerythrin (PE), BD Horizon V450, allophycocyanin, AlexaFlour647, PE-cyanin 7, or peridinin-chlorophyll protein conjugates. Dead cells were identified by staining with the fixable viability dye eFlour 780 (eBioscience, Frankfurt, Germany). Intracelluar staining for Foxp3 was performed with the Foxp3 staining kit (eBioscience, Frankfurt, Germany) according to the manufacturer’s recommendations. Cytokine production from freshly isolated splenocytes was measured by stimulating cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, München, Germany) and 100 µg/ml ionomycin (Sigma-Aldrich, München, Germany) for 4 or 6 h (IL-10), respectively, in the presence of 5 µg/ml Brefeldin A (for IFN-γ, TNF-α staining) and 5 µg/ml Monensin (for IL-10 staining), treating with 2% paraformaldehyde and 0.1% NP40, and staining with the respective antibody cocktail. Flow cytometric expression analyses were performed with an LSR II instrument using DIVA software (BD Biosciences, Heidelberg, Germany).
6
Quantifying Stem Cell Markers in iPSCs
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Control iPSC or met-IPSC colonies were collected using 1 mg/mL collagenase IV (Thermo Fisher Scientific, Waltham, MA, USA). A single cell suspension was obtained by incubation in Enzyme Free Cell Dissociation Buffer (Thermo Fisher Scientific, Waltham, MS, USA) followed by mechanical trituration. 100,000 cells were stained in 10 µL phosphate-buffered saline (PBS) supplemented with 1 µL of primary antibodies raised against SSEA4 conjugated to BD Horizon™ V450, SSEA3 conjugated to Phycoerythrin and TRA-1-60 conjugated to Alexa Fluor™ 647 (all of them from BD Biosciences, San Jose, CA, USA) for 30 min at 4 °C. Cells were then washed with PBS and analyzed using a MACSQuant flow cytometer (MiltenyiBiotec, Bergisch Gladbach, Germany).
7
Isolation and Analysis of Human Immune Cells
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Spleens were disaggregrated into RPMI 1640 medium supplemented with 2% FBS by grinding with plunger of a 3-mL sterile syringe (Cat. No. 309657, BD Biosciences, San Jose, CA, USA). The suspension was filtered through 70-μm cell strainers (Cat. No. 22-363-548, Thermo Fisher Scientific, Waltham, MA, USA). Bone marrow cells from the femora and tibiae were flushed into RPMI 1640 supplemented with 2% FBS using a 3-mL sterile syringe. Approximately 50 uL of blood was collected from each mouse via submandibular plexus route in an EDTA tube (Cat. No. 077051, RAM Scientific, Inc., Nashville, TN, USA). After red blood cells had been lysed with 1X BD Pharm Lyse™ lysing solution (Cat. No. 555899, BD Biosciences, San Jose, CA, USA), about 200,000 splenocytes, bone marrow cells, and white blood cells were stained with the mouse anti-human CD45 monoclonal antibody conjugated to BD Horizon™ V450 (Cat. No. 560367; BD Biosciences, San Jose, CA, USA) and mouse anti-human CD2 monoclonal antibody conjugated to APC (Cat. No. 300214; BioLegend, San Diego, CA, USA). Human CD45+CD2+ cells were analyzed on an Aurora flow cytometer (Cytek Aurora, Fremont, CA, USA).
8
Multiparameter Flow Cytometry Panel
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Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, allophycocyanin (APC)-, BD Horizon™ V450 (V450)-, and PE-Cy5-labelled anti-CD3 (UCHT1); APC-labelled anti-CD27 (L128); FITC-labelled anti-CD19 (HIB19); APC-labelled anti-CD45RO (UCHL1); APC-labelled anti-CD19 (HIB19); FITC-labelled anti-CD56 (MCAM16·2); Alexa Fluor® 700-labelled anti-CD4 (RPA-T4); APC-Cy™7-labelled anti-CD8 (SK1); APC-Cy™7-labelled anti-CD69 (FN50); FITC-labelled anti-CD95 (DX2); PE-Cy7-labelled anti-CD3 (SK7); PE-labelled anti-CD45RA (HI100); FITC-labelled anti-CD28 (CD28.2); FITC-labelled anti-CD94 (HP-3D9); FITC-labelled anti-T-cell receptor (TCR) αβ (WT31); PE-labelled anti-TCR αβ (T10B9.1A-31); FITC-labelled anti-CD69 (FN50); PE-Cy7-labelled anti-CCR7 (3D12); BD Horizon™ V500 (V500)-labelled anti-CD8 (RPA-T8); and 7-amino-actinomycin D (7-AAD) were purchased from BD Biosciences (Franklin Lakes, NJ). Pacific Blue™-labelled anti-CD107a (LAMP-1) was purchased from Biolegend (San Diego, CA). PE-labelled anti-TCR γδ (B1.1) was purchased from eBioscience (San Diego, CA). FITC-labelled anti-TCR pan γδ (IMMU510) was purchased from Beckman Coulter (Fullerton, CA). Pacific Orange-labelled anti-CD8 (3B5) was purchased from Invitrogen (Camarillo, CA).
9
Hypoxia Modulates T Cell Gene Expression
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PBMCs from HC cultured in 24-well plates with 1 mL of RPMI 1640 media supplemented 10% FBS, 200 mg/mL penicillin, 200 U/mL streptomycin, 4 mM L-glutamine were placed either in a hypoxia incubator, created by displacing O2 (2% O2) with infusion of N2 (93%), or a normoxic incubator (21% O2) for 12 h at 37°C. RNA was extracted using a Macherey Nagel kit according to the supplier’s recommendations. Complementary DNA was synthetized from 250 ng using a superscript III kit (invitrogen) and qPCR was performed to study TCF-7 and IL-7R expression. Finally, median fluorescence intensity was measured for CD127 (also called IL-7R) protein on CD3+CD4+ T cells by flow cytometry using the following antibodies (1/100e): CD3-PE-Cy7, CD4-PercP-Cy 5.5 and CD127-PE (BD, Biosciences). A Viability dye (BD Horizon V450, 1/1000e) may be used to exclude dead cells from analysis (LSR II BD Biosciences and FlowJo software).
10
Flow Cytometry Analysis of Dendritic Cell Markers
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Cell surface expression markers were examined using flow cytometry for HLA-DR,-DP,-DQ (FITC), and co-stimulatory molecules, CD40 (BD Horizon V450), CD80 (Alexa Fluor 700), and CD86 (allophycocyanin) using established protocols. 13 Briefly, eosinophils and CD4 + Th cells were removed from co-culture, washed and saturating quantities of primary antibodies or specific isotype controls were added to the cells and incubated for 40 min at 4°in the dark, washed and fixed. Human dendritic cells were identified by staining cells with CD1a (allophycocyanin) while eosinophils were identified by staining with a specific marker, siglec-8 (phycoerythrin). 26 An EBV-transformed B cell line was used as a source of cells that stained positively for HLA-DR/DP/DQ, CD40, CD80 or CD86 (see Supplementary material, Fig. S2). Multiple panels of conjugated antibodies were used to identify the subpopulation of immune cells and the corresponding specific cell surface markers. Ten thousand events were collected on a flow cytometer, LSRII (BD Biosciences, Oxford, UK) using FACS DIVA software (BD Biosciences, Oxford, UK). Analysis was performed using FLOWJO software (Treestar Inc., Ashland, OR). In all plots, dead cells, cellular debris and cell aggregates were excluded during the gating process.
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