Mice were transcardially perfused with PBS containing heparin, brains were dissected, collected in ice-cold PBS, and cut into small pieces. Tissue was digested in 1 ml Accutase (Merck) per brain at 37 °C for 30 min and triturated through 100 μm cell strainers which were rinsed with 10% FCS in PBS. Cells were purified by a linear 40% Percoll (GE Healthcare) centrifugation step at 650 g without brakes for 25 min and the myelin top layer and supernatant were discarded. Mononuclear cells were resuspended in FACS buffer (1% BSA and 0.1% sodium azide in PBS) and counted. Viable cells were identified by Live/Dead stain (Thermo Fisher), Fc receptors were blocked for 15 min with rat anti-CD16/32 (1:100, BD Pharmingen), and cells were washed and labeled with the following antibodies for 30 min at 4 °C: rat anti-CD11b APC (1:100, BioLegend), rat anti-CD45 FITC (1:100, BioLegend), rat anti-Siglec H PE (1:100, BioLegend). Cells were washed twice, single viable cells were gated, and analyzed using a FACSCalibur (BD Biosciences) and FlowJo (version 10) software.
Rat anti cd45 fitc
Manufactured by BioLegend
Rat anti-CD45-FITC is a lab equipment product that detects the CD45 antigen, which is expressed on the surface of all leukocytes. This antibody is conjugated with the fluorescent dye FITC, allowing for the visualization and identification of CD45-positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Rat anti cd49f pe, by BioLegend (3 mentions)
Rat anti cd326 epcam apc cy7, by BioLegend (3 mentions)
Goat anti trop2 apc, by R&D Systems (3 mentions)
Rat anti cd31 fitc, by BioLegend (3 mentions)
Rat anti ter119 fitc, by BioLegend (3 mentions)
Facsaria 2, by BD (3 mentions)
Y 27632 dihydrochloride, by Bio-Techne (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rat anti esam fitc, by BioLegend (2 mentions)
Rat anti cd44 fitc, by BioLegend (2 mentions)
Rpm1 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Corning (2 mentions)
Facscanto, by BD (2 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Rat anti cd16 32, by BD (1 mentions)
Ratanti cd16 32, by BioLegend (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Click it plus edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using rat anti cd45 fitc
1
Isolation and Analysis of Brain Mononuclear Cells
2
Cardiac Immune Cell Phenotyping
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Single cell suspensions were isolated from hearts 4 or 7 days after MI according to Percoll method. The cells were filtered through a 70 μm cell strainer and incubated with 1 μL / 1.0 × 106 cells rat anti-CD11b-APC/Cy7 (BioLegend, 101225), rat anti-CD45-FITC (BioLegend, 103107), rat anti-F4/80-PE (BioLegend 123109), rat anti-CD11b-PE (Biolegend,101207), rat anti-CD86-APC (BioLegend, 105007), rat anti-CD206-APC (BioLegend, 141707), and rabbit anti-Runx2 (Described above) antibodies on ice for 1 h after Fc receptor blockade by incubation with 1 μL / 1.0 × 106 cells rat anti-CD16/32 (BioLegend, 101301) antibody on ice for 30 min. FITC Rat IgG2a, κ Isotype Ctrl antibody (BioLegend, 400505), PE Rat IgG2a, κ Isotype Ctrl antibody (BioLegend, 400507), APC Rat IgG2a, κ Isotype Ctrl antibody (BioLegend, 400511), and APC/Cy7 Rat IgG2a, κ Isotype Ctrl antibody (BioLegend, 400523) were used for negative control antibodies. Intracellular Runx2 labeling was performed using FoxP3/Transcription Factor Staining Buffer set (Invitrogen, 00-5523-00) according to manufacturer’s protocol. Flow cytometric analysis was performed with FACS AriaII (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).
3
Cell Sorting and Antibody Staining
4
Cell Sorting and Antibody Staining
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Dissociated cells were stained with directly conjugated primary antibodies: rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend), goat anti-Trop2-APC (R &D Systems), rat anti-CD31-FITC (BioLegend), rat anti-CD45-FITC (BioLegend), and rat anti-Ter119-FITC (BioLegend) for 20 min on ice. Rat anti-ESAM-FITC (BioLegend) was also added to the Lin panel for some experiments. Rat anti-CD44-FITC (BioLegend) was used for analysis. Cells were stained in media containing RPM11640 (GIBCO), 10% FBS (Corning), 1x penicillin-streptomycin (GIBCO), and 10uM of the p160ROCK inhibitor Y-27632 dihydrochloride (Tocris Bioscience). Sorting was performed on a BD FACS Aria II (BD Biosciences) and flow cytometry analysis was performed on a BD FACS Canto (BD Biosciences).
5
Periosteal Cell Proliferation Assay
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Mice received 1.6 mg/kg EdU at 3 hr before sacrifice. Digested periosteal cells were stained with rat anti-Ter119 FITC (Biolegend, 116205), rat anti-CD31 FITC (Biolegend, 102509), rat anti-CD45 FITC (Biolegend, 147709), and rat anti-CD34 BV421 (BD Biosciences, 562608). EdU detection was carried out according to the manufacturer’s instructions (Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit, Thermo Fisher Scientific, C10424). Flow cytometry was performed by either LSR A or BD LSR Fortessa flow cytometer and analyzed by FlowJo v10.5.3 for MAC.
6
Prostate Cell Immunophenotyping by Flow
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Single-cell suspensions of 5 × 104–1 × 106 cells from mouse prostate tissue of different ages (each n = 4) were stained in cell staining buffer comprised of 1X DPBS (Gibco) with 5 g/L protease-free bovine serum albumin (Sigma-Aldrich) and 200 mg/L sodium azide (Sigma-Aldrich). Nonspecific antibody binding by Fc receptors was blocked using TruStain fcX (anti-mouse CD16/32) Antibody (BioLegend) according to the manufacturer’s protocol. Cells were then stained with rat anti-CD49f-PE (BioLegend), rat anti-CD326 (EpCAM)-APC/Cy7 (BioLegend), goat anti-Trop2-APC (R&D Systems), and rat anti-CD45-FITC (BioLegend) for 30 minutes at room temperature. Cells were washed with cell staining buffer then fixed in 1% paraformaldehyde (Electron Microscopy Sciences) for 10–15 minutes at 37°C. After fixation, cells were chilled on ice for 1 minute, washed with cell staining buffer, and stored at 4°C before analysis on a FACSCanto flow cytometer (BD Biosciences).
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