Cells were stained with monoclonal anti–mouse fluorescently conjugated antibodies: Sca-1 (D7), c-Kit (ACK2), CD3 (145-2C11), CD4 (GK1.5), CD8 (SK1), CD5 (53-73), CD19 (1D3), NK1.1 (PK136), CD11b (MI/70), CD11c (N418), CD16(32) (93), IgE (23G3), FcεRI (MAR-1), CD49b (DX5), CD115 (AFS98), F4/80 (BM8), and CD34 (RAM34) from eBioscience; or monoclonal anti–human fluorescently conjugated antibodies: CD19 (HIB19), FcεRI (AER-37), CD34 (4H11), c-Kit (104D2), and TCRαβ (IP26) from eBioscience. Samples were acquired on an LSRII or LSRFORTESSA X-20 flow cytometer (BD) and analyzed using FlowJo software (v10.0.5; Tree Star). Cell sorting was performed using a FACSAria II flow cytometer (BD). BM-resident and splenic progenitors were sort purified as CD3−, CD19−, CD11b−, CD11c−, NK1.1−, Sca-1−, FcεRI−, CD34+, and c-Kit+ cells. Splenic progenitors were isolated on day 7 after T. spiralis infection.
Cd34 ram34
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CD34 (RAM34) is a lab equipment product from Thermo Fisher Scientific. It is used to identify and enumerate CD34-positive cells, which are primitive hematopoietic stem and progenitor cells. The product's core function is to enable the detection and quantification of CD34-positive cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
C kit 2b8, by BioLegend (3 mentions)
Sca1 d7, by BioLegend (3 mentions)
B220 ra3 6b2, by BioLegend (2 mentions)
Cd150 tc15 12f12.2, by BioLegend (2 mentions)
Ter119 ter 119, by BioLegend (2 mentions)
Cd16 32 93, by Thermo Fisher Scientific (2 mentions)
Cd11b m1 70, by Thermo Fisher Scientific (2 mentions)
Cd31 390, by Thermo Fisher Scientific (2 mentions)
Cd45 30 f11, by BioLegend (2 mentions)
Gr 1 rb6 8c5, by BioLegend (2 mentions)
Cd45.1 a20, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa x 20 flow cytometer, by BD (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Cd4 rm4 5, by BD (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd41 mwreg30, by BD (1 mentions)
Il 7r a7r34, by Thermo Fisher Scientific (1 mentions)
Cd8a 53 6, by Thermo Fisher Scientific (1 mentions)
Ly 6g c rb6 8c5, by BioLegend (1 mentions)
Cd105 mj7 18, by BioLegend (1 mentions)
8 protocols using cd34 ram34
1
Flow Cytometric Analysis of Splenic Progenitors
2
Multiparametric Flow Cytometry Analysis
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All flow cytometric analyses and sorting were performed on a BD FACS Aria II cell sorter. Antibodies used were CD4 (RM4-5, BD Biosciences), CD8a (53-6.7, eBioscience), B220 (RA3-6B2, Biolegend), CD11b (M1/70, BD Biosciences), Ly6G/C (RB6-8C5, Biolegend), Ter119 (TER-119, Biolegend), Sca1 (D7, Biolegend), MPL (AMM2, Immuno-Biological Laboratories), CD41 (MWReg30, BD Biosciences), CD150 (TC15-12F12.2, Biolegend), cKit (2B8, Biolegend), CD34 (RAM34, eBioscience), IL7R (A7R34, eBioscience), Flt3 (A2F10, eBioscience), CD16/32 (93, eBioscience) and CD105 (MJ7/18, Biolegend). Streptavidin (eBioscience) was used to resolve biotinylated antibodies and propidium iodide was added prior to analysis to identify live cells.
3
Multiparametric Flow Cytometry of Immune Cells
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Antibodies against the following antigens were used: CD11b (M1/70; eBioscience), CD49b (DX5; eBioscience), Siglec-F (E50-2440; BD), Ly6G/C (RB6-8C5; BD), FcεRI (Mar-1; BioLegend), CD45 (30-F11; BioLegend), CD34 (RAM34; eBioscience), ESAM-1 (1G8; BioLegend), CD31 (390; eBioscience), c-kit (2B7; BioLegend), CD106 (429; eBioscience), IL-4Rα (mIL4-M1; BD), and CD213a1 (13MOKA; eBioscience). Key cell populations were defined as follows: (a) eosinophils, FSCloSSChiCD11b+Siglec-F+ or FSCloSSChiSiglec-F+4get+; (b) basophils, FSCloSSCloCD49b+FcεRI+Gr-1−CD4−CD8−CD19−γδ−Siglec-F− or FSCloSSCloBasoph8+CD49b+; (c) mast cells, 4get+ckit+ or FSChiSSChic-kit+CD11a−CD44+; (d) endothelial cells, CD45−CD34+ESAM-1+; and (e) PMNs/monocytes, Gr-1+CD11b+. Flow cytometry data acquisition was performed on an LSRII (BD), using FlowJo to analyze the data.
4
Multi-Parameter Cell Sorting and Analysis
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For cell sorting and analysis, monoclonal antibodies to CD41 (MWReg30, eBioscience), CXCR4 (2B11, eBioscience), CD11b (M1/70, eBioscience), F4/80 (BM8, eBioscience), Gr-1 (RB6-8C5, Biolegend), Ly6C (HK1.4, Biolegend), CD11c (N418, eBioscience), CD45.1 (A20, eBioscience), CD45.2 (104, Biolegend), CD4 (GK1.5, eBioscience), CD8 (53–6.7, Biolegend), INF-γ (XMG1.2, Biolegend), IL4 (11B11, Biolegend), CD34 (RAM34, eBioscience), Sca-1 (D7, Biolegend), c-kit (2B8, Biolegend), CD135 (A2F10, Biolegend), CD3ε (145–2 C11, Biolegend), CD45R (RA3-6B2, Biolegend), TER-119 (Ter-119, Biolegend), IgM (II/41, eBioscience), FγRII (93, Biolegend), IL-7R (A7R34, Biolegend), TNFα (MP6-XT22, Invitrogen), IL-6 (MP5-20F3, Biolegend), OVA257-264 (SIINFEKL) peptide bound to H-2Kb (eBio25-D1.16 (25-D1.16), Invitrogen) and IL-2 (JES6-5H4, eBioscience) were used where indicated.
5
Characterization of Enzymatically Isolated Human Skeletal Muscle Cells
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First, in order to characterize the enzymatically isolated cells and to determine the appropriate markers, FACS analysis was performed on freshly isolated human Sk-Cs using typical mesenchymal stem cell surface makers: CD29, CD31, CD44, CD56 (NCAM) and CD73 (purchased from BD Biosciences, Dan Jose, CA); CD105 and CD117 (c-kit) (from BioLegend, San Diego, CA); CD133 (from Miltenyi Biotec, Bergisch Gladbach, Germany); and CD166 (from Beckman Coulter, Brea, CA); as well as CD34 (RAM34; eBioscience, San Diego, CA) and CD45 (30-F11; BioLegend, San Diego, CA) antibodies. Cell analysis and sorting were carried out on a FACSAria (Becton Dickinson Japan, Tokyo, Japan). Note that the same cell characterization was also performed for expanded cells cultured under the conditions described blow. Consequently, based on the results for freshly isolated cells (see Figure 1A ), we decided to sort human Sk-Cs using CD29, CD34 and CD45, and obtained the CD34+/45−/29+ (Sk-34/29+), CD34+/45−/29− (Sk-34/29−), CD34−/45−/29+ (Sk-DN/29+), and CD34−/45−/29− (Sk-DN/29−) fractions (see Figures 1B,C ).
6
Monoclonal Antibodies for Cell Identification
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The monoclonal antibodies used in this study targeted the following proteins: CRLR (H-42; Santa Cruz, Dallas, TX, USA), RAMP-1 (EPR10867; abcam, Cambridge, UK), Gr-1 (RB6-8C5; BioLegend, San Diego, CA, USA), Mac-1 (M1/70; BioLegend), B220 (RA3-6B2; BioLegend), CD3e (145-2C11; Biolegend), CD2 (RM2-5; TONBO, Japan), CD8a (53–6.72; TONBO), TER-119 (TER-119; TONBO), CD127 (A7R34; TONBO), c-Kit (2B8; BioLegend), Sca-1 (D7; BioLegend), CD34 (RAM34; eBioscience, Santa Clara, CA, USA), CD16/32 (93; eBioscience), CD48 (HM48-1; BioLegend), CD150 (TC15–12F12.2; BioLegend), CD45.2 (104; eBioscience) and CD45.1 (A20; eBioscience). A mixture of mAbs against CD2, CD3e, CD8a, B220, TER-119, CD127, Mac-1 and Gr-1 served as lineage markers (Lineage). Mouse anti-BrdU-Alexa Fluor 647 antibody (3D4; BD Biosciences) was used to detect intracellular BrdU.
7
Immunohistochemical Analysis of Lung Tissue
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Formalin-fixed and paraffin-embedded lungs were cut into 5-µm sections for Masson's trichrome or hematoxylin and eosin staining. For immunostaining, lung sections underwent antigen retrieval and were stained using antibodies against CD34 (RAM34) (eBiosciences), podocalyxin (AF1556) (R&D Systems), GFP (ab13970) (Abcam), vimentin (ab92547) (Abcam), surfactant protein C (AB3786) (Millipore), E-cadherin (36/E-cad) (BD), and keratin 5 (Poly9059) (Biolegend). Sections were then incubated with AlexaFluor-conjugated antibodies and mounted using Prolong Gold Antifade with DAPI (Life). Optical z-stack images were captured on a Leica SP5X confocal microscope and morphometric analysis was performed using ImageJ.
8
Multiparameter Flow Cytometry Analysis of Lung Cells
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BALF was collected by three tracheal instillations and aspirations of 1 mL PBS. Tissues were digested with collagenase D (1.5 U/mL) and dispase II (2.4 U/mL) (Roche) for 30 minutes. Samples were then incubated with anti-CD16/32 to block nonspecific antibody binding. Fluorescence-conjugated antibodies to CD45 (I3/2), CD11c (N418), CD3e (145-2C11), CD8 (53.67), CD4 (GK1.5), B220 (RA3-6B2), Ly6B (7/4) (Abcam), SiglecF (E50-2440) (eBiosciences), CD34 (RAM34) (eBiosciences), CD31 (390) (eBiosciences), PDGFRα (APA5) (eBiosciences), Sca1 (D7) (eBiosciences), and EpCAM (G8.8) (eBiosciences) were used. For EdU uptake experiments, mice were given 1 mg EdU daily by intraperiotenal injections; EdU detection was performed using the Click-IT assay kit (Life). Data was acquired on a BD LSRII and analyzed with FlowJo Software.
All antibodies were generated in-house (UBC AbLab) unless otherwise indicated.
All antibodies were generated in-house (UBC AbLab) unless otherwise indicated.
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