Human BMSC and BC cell lines 5637 and T24 were all offered by the American Type Culture Collection (ATCC, USA). Both BC cells and BMSC were cultured in DMEM (Hyclone, USA), supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and penicillin/streptomycin (100 U/mL, Gibco, USA). To identify BMSC, the BMSC were collected and re-suspended on PBS, adjusting the cell density to 2 × 105 cells/mL. Then, 10 μL CD29-PE, CD44-PE, CD106-FITC, CD34-FITC, or CD45-FITC antibodies (Invitrogen, USA) were added into 200 μL cell suspension and allowed for 30 min incubation in dark. Cell suspension was then centrifuged for 5 min at 1000 g. The sediment was re-suspended in PBS, and the expression levels of these biomarkers were determined by flow cytometry (FACSCalibur, BD Biosciences, USA).
Cd44 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD44-PE is a fluorescently-labeled antibody that binds to the CD44 cell surface receptor. It is used for the identification and analysis of cells expressing the CD44 antigen in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd34 fitc, by Thermo Fisher Scientific (5 mentions)
Facscalibur, by BD (5 mentions)
Cd62l fitc, by Thermo Fisher Scientific (5 mentions)
Cd90 pe, by Thermo Fisher Scientific (5 mentions)
Cd45 fitc, by BD (5 mentions)
Cd45 pe, by Thermo Fisher Scientific (5 mentions)
Cd29 pe, by Thermo Fisher Scientific (4 mentions)
Cd31 pe, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Cd62l apc, by Thermo Fisher Scientific (3 mentions)
Cd8 percp cy5, by Thermo Fisher Scientific (3 mentions)
Cd4 apc, by Thermo Fisher Scientific (3 mentions)
Cd11b pe, by Thermo Fisher Scientific (3 mentions)
Cd105 apc, by Thermo Fisher Scientific (3 mentions)
Cd105 pe, by Thermo Fisher Scientific (3 mentions)
Cd4 fitc, by Thermo Fisher Scientific (3 mentions)
Cd8 pe cy7, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (3 mentions)
Cd106 fitc, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
44 protocols using cd44 pe
1
Characterization of BMSC and BC Cells
2
Expansion and Characterization of hMSCs
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The hMSCs passaged for four times were counted for cell numbers and adjusted for cell concentration for preparation of 1 × 106/ml cell suspension. Cell morphologies were observed under a microscope. Cell suspensions were inoculated at 6-well plates with 2 ml for each well. In total, 24 wells were inoculated for incubation at 37 °C with 5% CO2. The culture medium was replaced every 48 h. Cells were digested by pancreatin at an interval of 24 h. Cells from three duplicate wells were counted for continuously 8 days to calculate the average cell numbers and to draw cell growth curve. After that, 100 μl of cell suspension was added into an Eppendorf tube, in which each 10 μl of CD29-PE, CD44-PE, CD106-FITC, CD34-FITC, or CD45-FITC (Invitrogen, USA) was added and mixed for incubation at room temperature for 30 min. Another Eppendorf tube with cell suspension was set as blank control. PBS washing was performed before the Eppendorf tube was centrifuged at 1000g for 5 min. Sediment was re-suspended using PBS. The expressions of exosomal biomarkers, CD29, CD44, CD106, CD34, and CD45 were determined using flow cytometry (FCM).
3
Quantification of TrxR1, CD44, E-Cadherin, and N-Cadherin Levels in Cells
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Cells were seeded at a density of 2 × 104/well on 4-well Lab-Teck II chamber slides (Nunc, Naperville, IL, USA). After indicated treatment, cells were washed twice with ice-cold PBS and fixed immediately with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After two washes with PBS, cells were permeabilized with 0.25% Triton X-100 for 10 minutes at room temperature. Then cells were blocked with 1% bovine serum albumin (BSA) in PBS/Tween 20 for 30 minutes at room temperature and further incubated overnight with the primary antibodies against TrxR1 (Abcam, 1:300), CD44-PE (eBioscience, 1:160), E-Cadherin (BioLegend, 1:300) or N-Cadherin (Biolegend, 1:300) diluted in 1% BSA in PBS/Tween 20 in a humidified chamber at 4 °C. After three rinses with PBS, secondary antibodies conjugated with Dylight 488, Dylight 649 or Dylight 549 (Abbkine, 1:300) diluted in 1% BSA in PBS/Tween 20 (1:200) was added and incubated for 1 hour at room temperature in dark followed by three washes with PBS. Subsequently, nucleus were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) for 10 minutes. Images were taken with a laser scanning confocal microscope (Nikon A1, Japan) and analyzed by NIS-Elements Viewer 4.20 software.
4
Lung Cell Isolation and Immunophenotyping
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Single-cell suspensions of the lung tissues were prepared by cutting them into small pieces followed by incubation in Dulbecco’s Modified Eagle Media (DMEM) containing 0.18 mg/mL Collagenase Type I (Sigma, St. Louis, MO, USA), 0.02 mg/mL DNase I (Sigma, St. Louis, MO, USA) for 1 h at 37 °C under constant rotation, followed by being mechanically passed through a 100 μm and 70 μm cell strainer sequentially. Erythrocytes were lysed using RBC lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). Cells were then counted and subjected to flow cytometry. Lymphoid and myeloid compartments were investigated in the lung samples of mice on various intervention diets. Antibodies used for flow cytometry analysis were as follows: CD64-PeCy7 (Clone X54-5/7.1), Ly6C-PerCPCy5.5 (Clone AL-21), CD11b-V450 (Clone M1/70), MHCII-APC (Clone M5/114.15.2), CD103-PE (Clone M290), CD11c-A700 (Clone HL3), SiglecF-APCCy7 (Clone E5-2440), Ly6G-FITC (Clone 1A8), PD-1-FITC (Clone 29F.1A12), CD4-BV510 (Clone RM4-5), CD44-PE (Clone IM7), NK1.1-APCCy7 (Clone PK136), CD3-A700 (Clone 500A2), CD62L-V450 (Clone MEL-14), CD19-PerCPCy5.5 (Clone 1D3), CD8-APC (Clone 53-6.7), and KLRG1-BV786 (Clone 2F1) purchased from BD (Biosciences, Johannesburg, SA) and eBioscience (ThermoFisher, Johannesburg, SA) [29 (link),30 (link)].
5
Immunostaining of Decalcified Murine Bones
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Mice were euthanized at the indicated ages, and perfused with 4% PFA. The ulna, radius, femur, and tibia of each animal were collected, placed into 4% PFA for 2 h, and then washed with PBS. Bones were decalcified as previously described by placing the bones in 0.5 M EDTA for 24 h, then prepared for mounting by incubation in sucrose for 24 h29 (link). Long bones were flash frozen in OCT (Fischer Healthcare), sectioned onto adhesive tape as previously described30 (link), washed, blocked with 1% rat serum in PBS, and then stained. Staining antibodies used included: DAPI, TCRβ-PE (Clone: H57-597, eBioscience), and CD44-PE (Clone: IM7, eBioscience). After staining, the tissues were mounted with Fluoromount-G (Invitrogen) and imaged using a Zeiss Axiovert 200 M Fluorescence Microscope (Zeiss) in the Leduc Imaging Core (Brown University). Images were analyzed with FIJI31 (link).
6
Flow Cytometry Analysis of Immune Cell Populations
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BMNC. PECs, lymph or splenic cells were washed in FACS buffer, blocked for 10 min in Fc block and incubated with various conjugated antibodies. To determine antigen specific T cells: CD3-FITC, OTI-CD8-dsRed, MHC I-Ova dextramer-APC (Immudex, Denmark and Tetramer core facility NIH, Bethesda, MD); to determine T cell activation: CD3-PerCp (145-2C11), CD4-PECy7(RM4-5), CD8-FITC (53-6.7), CD44-PE (IM-7), CD62L-APC (MEL-14); to determine monocyte populations: CD11c-APCCy7 (N418), CD11b-FITC (M1/70), Ly6C-PE (AL-21), Ly6G-APC (RB6-8C5), MHCII-PECy7 (M5/114.15.2), and CD45-PerCp (30F11), (eBioscience, San Diego, CA). Cells were analyzed using a BD FACSCanto II flowcytometer (BD Biosciences, San Jose, CA) and FlowJo analysis software v.8.7.3 (Treestar Software, Ashland, OR). Cell populations were determined by gating on CD4+, CD8+, (T cells) CD45hi/CD11b+ (macrophages) and CD45int/CD11b+ (microglia) Ly6CintLy6G+, (neutrophils) Ly6C+Ly6G−, (inflammatory monocytes) from a live cell gate (gating strategy as depicted in Supplementary Figs. 5 , 7 ).
7
Multi-Dimensional Flow Cytometry Profiling
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Splenocytes, mesenteric, and inguinal lymph node cells were isolated and then stained with anti-mouse CD4-APC, CD8-PerCP-Cy5.5, CD44-PE, CD62L-FITC, GL-7-FITC, CD95-PE, and B220-PerCP-Cy5.5 antibodies (eBioscience, San Diego, CA) for 15 min at 4 °C. For TFH differentiation analysis, the cells were stained with anti-mouse CXCR5-biotin for 30 min at 4 °C followed by anti-mouse CD44-FITC, CD4-PerCP-Cy5.5, and streptavidin-APC staining. After fixation and permeabilization using the Foxp3 Staining Kit (eBioscience), anti-mouse Bcl-6-PE was stained for 1 h at room temperature. Cells were examined using the FACSCanto II system (BD Bioscience, San Jose, CA, USA) and data were analyzed using Flow Jo software (Treestar, Ashland, OR, USA). In all cases, doublets (FSC-area versus FSC-height gating) were excluded.
8
Evaluating the Impact of SPIONs Clusters on ADSC Phenotypes
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To investigate the effect of SPIONs clusters@PDA on cell phenotypes of ADSCs, cells were seeded at a density of 1 × 105 cells/well and allowed to attach for 24 hrs. Next, cells were treated with or without 0.25 mM SPIONs clusters@PDA for another 24 hrs. Then, the unlabeled and labeled cells were trypsinized and suspended with PBS containing 5% bovine serum albumin (BSA, Sigma, USA); afterwards, the cells were incubated with different primary antibodies as indicated for 60 min at room temperature, including CD29-PE and CD44-PE (eBioscience, CA), CD31, CD34, CD45, CD73 and CD90 (Santa Cruz Biotechnology, USA), as well as CD105 and HLA-DR (Abcam, UK), respectively. Subsequently, the cells were washed with PBS for twice, and then incubated with the corresponding fluorescent conjugated secondary antibodies (donkey anti-mouse IgG- Alexa fluor 488 or donkey anti-rabbit IgG-alexa fluor 647, Invitrogen, USA) for 30 min at room temperature. Finally, the cells were characterized by flow cytometry.
9
Flow Cytometric Analysis of Stem Cell Markers
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The cultured cells were detached from the dish by adding a solution of 0.25% trypsin and 0.02% EDTA (Gibco, USA), and then by washing with PBS. A cell pellet was collected after centrifugation at 1,500 rpm for 3 min and then resuspended in test tubes containing PBS (1×106 cells/ml). Fluorescence conjugated antibodies, CD24-PE, CD44-PE, and CD133-FITC (eBioscience, USA), were added to single cells for 30 min at 4°C. All samples were washed with PBS, and cells were fixed in 1% paraformaldehyde. Phenotypic analysis was performed with a flow cytometer (FACSCalibur, BD Biosciences, USA).
10
Phenotyping of Mesenchymal Stem Cells
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In order to identify the mesenchymal stem cell phenotype, approximately 5 × 105 PDLSCs were incubated in 5% bovine serum albumin (BSA)/phosphate buffered saline (PBS) (Gibco, Invitrogen, Carlsbad, USA) 1 × at 4 °C in dark for 1 h with the following monoclonal antibodies: PAR1-FITC (Abcam, Cambridge, UK), OCT4-FITC (Abcam, Cambridge, UK), SOX2-FITC (Abcam, Cambridge, UK), STRO-1-FITC (Abcam, Cambridge, UK), CD14-FITC (eBioscience, San Diego, USA), CD90-FITC (eBioscience, San Diego, USA), CD31-PE (eBioscience, San Diego, USA), CD-44-PE (eBioscience, San Diego, USA), CD34-FITC (Biolegend, San Diego, USA) and CD146-PE (Biolegend, San Diego, USA) for 30 min at 4 °C. Unstained control was used to set gates. A total of 10–50,000 events were recorded and data analyzed through FlowJo (Becton Dickinson, Oregon, USA).
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