Prior to perfusion, spleens were harvested from experimental mice. Single cell suspensions from spleens were treated with ACK erythrocyte lysis buffer. Mice were perfused with 25 mL of ice-cold PBS and brains and spinal cords were collected from perfused mice and dounce homogenized to obtain single cell suspensions. CNS cells were purified by centrifugation for 30 min in a 30% Percoll (GE Healthcare) solution as previously reported (25 (link)). Cells were incubated with the anti-Fc receptor antibody 2.4G2 prior to the addition of antibodies. The following antibodies were purchased from BD Biosciences: CD45-FITC, CD8α-APC-H7, CD19-APC-H7, CD19-BV510, B220-PE-TxRed, B220–PE-CF594, CD11b-AlexaFluor-700, MHC-v450. The following antibodies were purchased from eBioscience: MHCII-Pacific Blue, CD11c-PECy7. The following antibodies were purchased from BioLegend (San Diego, CA): CD138-PE, MHCII (I-A/I-E)-Pacific Blue, Thy1.1-PerCP, CD4-APC. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) and analyzed with FloJo software (TreeStar) with doublets being excluded.
Cd19 apc h7
Manufactured by BD
Sourced in United States, Germany, United Kingdom
CD19-APC-H7 is a fluorescent-conjugated antibody that binds to the CD19 antigen. It can be used for the identification and enumeration of CD19-positive cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Cd8 percp, by BD (3 mentions)
Cd45 fitc, by BD (2 mentions)
Cd33 bv510, by BD (2 mentions)
Compbead, by BD (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Mouse anti human gpr15 antibody, by R&D Systems (2 mentions)
Anti cd3 fitc, by Beckman Coulter (2 mentions)
Facs lysing solution, by BD (2 mentions)
Cd4 bv510, by BD (2 mentions)
Cd45 v500, by BD (2 mentions)
Cd10 apc, by BD (2 mentions)
Facsaria 2, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Cd24 pe, by BD (2 mentions)
Igd fitc, by BD (2 mentions)
Cd8 pe, by BD (2 mentions)
Cd27 pe, by BD (2 mentions)
Cd11b alexa fluor 700, by BD (1 mentions)
28 protocols using cd19 apc h7
1
Immune Cell Isolation and Analysis
2
Multiparametric Flow Cytometry of tPCLS
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Single‐cell suspensions of tPCLS were generated as described above. Single‐cell suspensions were blocked with FcR blocking reagent (Miltenyi Biotec) in 0.5% PBS‐BSA for 20 min, stained with fluorochrome‐conjugated antibodies, and analyzed on a FACSSymphony A5SE flow cytometer (BD Biosciences). Live single cells were identified by FSC and SSC characteristics. The data were analyzed using FlowJo V10 (TreeStar). All antibodies and secondary reagents were titrated to determine optimal concentrations. Comp‐Beads (BD) were used for single‐color compensation to create multicolor compensation matrices. For gating, fluorescence minus one control was used. The instrument calibration was controlled daily using Cytometer Setup and Tracking Beads (BD Biosciences). The following antibodies were used: CD3‐BUV805 (#612896, BD Biosciences), CD4‐BB630 (#562316, BD Biosciences), CD8‐BV650 (#743067, BD Biosciences), CD14‐PerCP‐Cy5.5 (#561116, BD Biosciences), CD15‐BUV805 (#742057, BD Biosciences), CD16‐BV650 (#563692, BD Biosciences), CD19‐APC‐H7 (#560252, BD Biosciences), CD25‐PE‐Cy7 (#557741, BD Biosciences), CD33‐BV510 (#563257, BD Biosciences), CD45‐AF700 (#368514, BD Biosciences), CD80‐BV711 (#740801, BD Biosciences), CD206‐PE/Cy7 (#321124, BioLegend), CD326‐FITC (#324203, BioLegend), HLA‐DR‐APC/Fire750 (#307658, BioLegend), MerTK‐BV421 (#367603, BioLegend).
3
GPR15 Expression on Lymphocyte Subsets
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GPR15 surface expression concomitant with markers of differentiation on lymphocytes was analysed by flow cytometry. Briefly, 100 μl of blood specimen was incubated with mouse-anti-human-GPR15 antibody (1:500; R&D Systems, Wiesbaden-Nordenstadt, Germany) supplemented with 5% goat serum for 1 h. After washing in PBS/1% fetal calf serum (FCS) the GPR15 was stained with R-phycoerythrin-labelled goat-anti-mouse IgG2b (1:500, 1 h; Biozol, Eching, Germany) following an additional wash step. Thereafter, cells were incubated with 5% mouse serum for 30 min following a double-staining step for leucocyte differentiation markers (1 h; anti-CD3-FITC [Beckman Coulter, Krefeld, Germany], − CD4-BV510, −CD8-PerCP, −CD19-APC-H7 [BD Biosciences, Heidelberg, Germany], −CD16-PerCP, −CD56-PerCP [EXBIO, Prague, Czech Republic]). At the end, erythrocytes were lysed in FACS Lysing solution (BD Bioscience, Heidelberg, Germany) according to manufacturer’s instruction immediately before measurement. All measurements were performed on a FACS Canto II and analysed with the BD FACS DIVA software (version 8.0.1, BD Biosciences, Heidelberg, Germany).
4
Phenotypic Analysis of Cell Suspensions
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Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1 ). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table ; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 106 cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH4Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 104 cells were acquired in the CD19+ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).
Samples containing 106 cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH4Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 104 cells were acquired in the CD19+ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).
5
Lymphocyte Isolation and Characterization
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Lymphocytes were isolated as described and stained with anti-mouse IgD PerCP-Cy5.5 (1:400, 405710), LPAM (integrin α4β7) PE (1:100, 120605; (Biolegend, San Diego, CA), B220 V500 (1:400, 561227), CD19 APC-H7 (1:200, 560143), CD80 PE (1:500, 553769), CD273 APC (1:200, 560086), CD138 PE (1:200, 553714), IgM PE-Cy7 (1:200, 552867; BD Biosciences), CCR9 PE-Cy7 (1:100, 25-1991), CD73 PE-Cy7 (1:50, 25-0731), IgA PE (1:50, 12-4204), GL7 eFluor 450 (1:100, 48-5902), CD38 Alexa700 (1:800, 56-0381), CD21/35 Pacific Blue (1:800, 57-0212; eBiosciences) or CCR10 APC (1:100, FAB2815A; R and D systems. Minneapolis, MN) and were analysed using LSR II (BD Biosciences) or Navios (Beckman Coulter, Brea, CA) flow cytometers. For sorting, cells were labelled with anti-mouse CD138 PE (1:200, 553714), CD19 PE-Cy7 (1:200, 552854), CD80 APC (1:200, 560016) and GL7 FITC (1:100, 553666) before sorting using a FACSAria (BD Biosciences). Cells were sorted into tubes that had been coated with 2% BSA/PBS overnight, and pelleted by centrifugation at 600 g before being resuspended in PBS and injected into recipient mice or used for gene sequence analysis.
6
Multiparameter Flow Cytometry for B-ALL MRD Detection
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MPFC was performed with a panel of antibodies designed for B‐ALL. The diagnostic panel for B‐ALL contained CD45, CD7, CD19, CD13, CD33, CD34, CD117, HLA‐DR, CD10, cMPO, cCD3 and cCD79a and its extended panel included CD66c, CD22, CD20, CD58, CD38, CD123, CD45, cytoplasmic heavy chain of immunoglobulin M(cu) and cytoplasmic TDT (BD Bioscience; San Jose, CA; Beckman‐Counter; Indianapolis, IN).
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
15 (link) FACS Aria II (BD Biosciences) with Diva program was used to acquire and analyze the data. MRD panel included CD58‐FITC, KORSA‐PE, CD34‐Percp, CD20‐PE‐cy7, CD10‐APC, CD19‐APC‐H7, CD38‐V450 and CD45‐V500 (BD Bioscience and Beckman‐Counter).
Negative MRD by MPFC was defined as <10−4 blasts (0.01%) in bone marrow samples and Flow‐MRD positivity was defined as >10−4 blasts (0.01%) in bone marrow. In our study, patients with sustained undetectable MRD were defined according to negative MRD results observed at the end of consolidation and any of the previous time points.
7
Multicolor Flow Cytometric Analysis
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Blood was washed in PBS, stained with live/dead fixable blue dead cell staining kit (Invitrogen), washed in PBS and blocked in 2% FBS-PBS / 0.02% sodium azide plus Fc-block (Anti-CD16/32 antibody 1:200, BD Biosciences). Surface antigens were detected by incubation for 30 min at 4°C with conjugated antibodies including CD45.1-eFluor 450 (eBioscience Cat# 48-0453-82, RRID:AB_1272189 ), CD45.2-FITC (BD Biosciences Cat# 553772, RRID:AB_395041 ), CD3e-APC (BD Biosciences Cat# 561826, RRID:AB_10896663 ), CD19-APC-H7 (BD Biosciences Cat# 560143, RRID:AB_1645234 ), CD11b-PE (BD Biosciences Cat# 562287, RRID:AB_11154216 ) and GR1-Alexa Fluor 700 (BioLegend Cat# 108422, RRID:AB_2137487 ). Following washing with 2% FBS-PBS 0.02% sodium azide, red cells were lysed and leukocytes fixed by incubating in lyse/fix solution (BD Biosciences). Cells were washed with PBS and analyzed on an LSR-II cytometer (Becton Dickenson). Data were processed using FlowJo Software (Tree Star).
8
Multiparametric Cell Immunophenotyping
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Aqua Viability Dye (AqVi; Invitrogen) exclusion was used to identify viable cells. The antibodies used for these studies were: CD3-ECD (Beckman Coulter), CD8-APC (BD Pharmingen), CD19-APCH7 (BD Biosciences), HIV-1 p24-PE (Beckman Coulter), IL-17-V450 (BD biosciences), IFN-γ-AF700 (BD biosciences) and AnnexinV-Pacific Blue (Life Technologies). Isotype controls were used to establish the gates for IL-17 (Mouse IgG1- V450, BD Biosciences) and IFNγ (Mouse IgG1-AF700, BD biosciences).
9
Immunophenotyping of PBMC subsets following VitD3 treatment
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To identify specific immune cell subsets in PBMCs following VitD3 treatment, cells were stained with fluorescently-conjugated monoclonal antibodies; CD4-BUV737, CD45RO-APC, CD161-FITC, CD194-V450, CD196-PE, CD14-BV605, CD19-APC-H7, CD56-BV421, CD282-AF647 (TLR2), CD284-BV786 (TLR4; all from BD Bioscience; San Diego, CA, USA), and anti-TLR7-PE (Gibco Life Technologies, Carlsbad, USA), Zombie Aqua™ Fixable Viability Kit (BioLegend, San Diego, USA). Compensation bead particles were used to account for spectral overlap (BD Bioscience, San Diego, CA, USA) and analyzed using the BD LSRII flow cytometer. Unstained PBMCs and fluorescence minus one (FMO) were used as controls and a minimum of 100,000 events were analyzed per sample gated on live, single cell lymphocyte gate based on FSC and SSC, where the expression of the cell surface molecules was evaluated using FlowJo, LLC v10.4.2 software. Refer to Supplementary Figures 1–4 for gating strategies.
10
Phenotyping of Immune Cells in Periodontitis
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PBMCs and periodontal-infiltrating leukocytes from enrolled subjects were surface-stained with anti-human CD3-PerCP (BD Biosciences, San Jose, CA), CD19-APC-H7 (BD Pharmingen), NKp46-PE-Cy7 (BD Pharmingen), and CD14-FITC (eBioscience, San Diego, CA), and were intracellularly stained with anti-human IL-18 (Abcam), which was conjugated by PE/R-Phycoerythrin Conjugation Kit (Abcam). PBMCs, splenocytes, and periodontal-infiltrating leukocytes from mice were surface-stained with anti-mouse CD3-FITC, CD4-APC, CD8-PerCP Cy5.5, NK1.1-PE, CD11b-FITC, CD11c-PE, CD19-APC, and/or Ly-6G (Gr-1)-PE Cy7 (eBioscience) for detection of leukocyte subsets. Periodontal ligament cells were stained with Annexin V-FITC and propidium iodide. Data were acquired using FACS Aria II flow cytometer (BD Biosciences) and were analyzed using FlowJo Version 10 (Tree Star, Ashland, OR).
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