For intravascular staining, mice were intravenously injected with 8 μg of CD45 PerCPCy5.5 (clone 30-F11, Biolegend 103132) diluted in 200 μL of PBS. After 5 min, mice where euthanized, thymus and blood where collected. Blood was collected in a 20 mM EDTA coated tube and red blood cells were lysated with ACK lysis buffer prior to cell staining (as described above). Thymus was collected in MACS buffer (PBS, 2% FBS, 5 mM EDTA) in presence of 1.25 μg of CD45 unconjugated antibody (clone 30-F11, Biolegend 103101). A single cell suspension was prepared by smashing the organ prior to cell staining.
Cd45 percpcy5.5 clone 30 f11
Manufactured by BioLegend
Sourced in Germany
CD45 PerCPCy5.5 (clone 30-F11) is a fluorochrome-conjugated antibody that binds to the CD45 protein, which is expressed on the surface of various leukocyte populations. It can be used for the identification and immunophenotyping of immune cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Dispase 2, by Worthington (1 mentions)
Cd11b apc cy7 clone m1 70, by BioLegend (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Efluor 450, by Thermo Fisher Scientific (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Dnase 1, by Worthington (1 mentions)
Ghost dyetm red 710, by Cytek Biosciences (1 mentions)
Cytofix fixation buffer, by BD (1 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Ccr2 bv421, by BioLegend (1 mentions)
Mhcii apc cy7, by BioLegend (1 mentions)
Ly6g pe cy7, by BioLegend (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
4 protocols using cd45 percpcy5.5 clone 30 f11
1
Intravascular Immune Cell Labeling
2
Skin Leukocyte Isolation and Analysis
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Three 6 mm punch biopsies from each animal were performed and tissues pooled for processing and staining. Skin specimens were digested using 2 mg ml−1 Dispase II, 2 mg ml−1 Collagenase Type IV (Worthington Biochemical, Lakewook, NJ), 200 U ml−1 DNase I (Worthington Biochemical, Lakewook NJ), 2 mM Ca2+Cl2 and 2 mM Mg2+Cl2 for 45 min at 37 °C. Single-cell suspensions were counted in a Neubauer cell chamber and stained with the following antibodies (all from Biolegend, San Diego, CA): CD45-PerCP/Cy5.5 (clone 30-F11), CD11b-APC/Cy7 (clone M1/70), Ly6G-FITC (clone A1-8) and Siglec F-PE (clone E50-2440). Nonspecific staining was prevented with CD16/CD32 blockade (clone 24G2). Dead cells were excluded using the fixable viability dye eFluor780 or eFluor450 (eBiosciences, San Diego, CA). Flow analysis was performed on an LSR-II Flow Cytometer and analysed using Flow Jo Software. Total number of cellular infiltrates was calculated from relative frequencies.
3
Paw Cell Isolation and Characterization
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Paws were digested as described above, cell isolates were stained witn Ghost Dye TM Red 710 (Tonbo, San Diego, USA) according to the manufacturer´s recommendation and fixed with BD Cytofix TM Fixation buffer. Samples were stained after blocking of the FcR using mouse FcR blocking reagent (Miltenyi Biotec, Bergisch-Gladbach, Germany) with the following antibodies: CD45-PerCP/Cy5.5 (clone 30-F11, Biolegend), CD31-BV510 (clone 390, BD Bioscience), CD90. unmixing on a Cytek NL-3000 and data were analysed using SpectroFloR V3 (Scytek Biosciences, Freemont, USA) and FlowJo V10 (BD Biosciences).
4
Isolation and Characterization of Cardiac Immune Cells
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Single-cell suspensions were generated from saline perfused hearts by finely mincing and digesting them in DMEM with Collagenase 1 (450 U/mL), Hyaluronidase (60 U/mL), and DNase I (60 U/mL) for 1 h at 37°C. All enzymes were purchased from Sigma. To deactivate the enzymes, samples were washed with HBSS that was supplemented with 2% FBS and 0.2% BSA and filtered through 40 μM cell strainers. Red blood cell lysis was performed with ACK lysis buffer (Thermo Fisher Scientific). Samples were washed with HBSS and resuspended in 100 μL of FACS buffer (DPBS with 2% FBS and 2 mM EDTA). Cells were stained with monoclonal antibodies at 4°C for 30 min in the dark. All the antibodies were obtained from Biolegend: CD45-PerCP/Cy5.5, clone 30-F11; CD64-APC and PE, clone X54-5/7.1; CCR2-BV421, clone: SA203G11; MHCII-APC/Cy7, clone M5/114.15.2; Ly6G-PE/Cy7, clone 1A8; and Ly6C-FITC, clone HK1.4. Samples were washed two times, and final resuspension was made in 300 μL FACS buffer. DAPI or LIVE/DEAD™ Aqua dyes were used for the exclusion of dead cells. Flow cytometric analysis were performed on the BD Fortessa platform. Neutrophils were gated as CD45 + Ly6Ghigh. Macrophages were gated as Ly6GnegCD64highLy6Clow cells.
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