1 × 106 cells were Fc blocked (Biolegend101320) and stained for 1 hour, on ice, in the dark, as previously described.20 (link) The following antibodies were used, B220−FITC (BD553087), I Ab PerCP−Cy5.5 (Biolegend116416), Ly6C PE or PECy7 (Biolegend128008, 128071), HLA−DR PECy7 (eBioscience25−9956−41/Biolegend307606), CD11b BV510 (BD562950/Biolegend101263), Ly6G BV605 (BD563005/Biolegend127639), CD11c APC (BD550261), CD3 A700 (eBioscience56−0032−82/Biolegend100216), Live Blue Fluorescent reactive dye (LifeTech L34962), and CD45 BV650 (Biolegend103151).
Cd45 bv650
Manufactured by BioLegend
Sourced in United States
CD45-BV650 is a fluorochrome-conjugated antibody that binds to the CD45 protein, a common leukocyte antigen expressed on the surface of most hematopoietic cells. It is commonly used in flow cytometry applications for the identification and enumeration of various immune cell populations.
Automatically generated - may contain errors
Lab products found in correlation
O4 pe, by Miltenyi Biotec (3 mentions)
Cd11b bv421, by BioLegend (2 mentions)
Fortessa flow cytometer, by BD (2 mentions)
Kaluza v2, by Beckman Coulter (2 mentions)
Glast apc, by Miltenyi Biotec (2 mentions)
Cd3 a700, by Thermo Fisher Scientific (1 mentions)
Fc blocked, by BioLegend (1 mentions)
Hla dr pecy7, by Thermo Fisher Scientific (1 mentions)
B220 fitc, by BioLegend (1 mentions)
Ly6g bv605, by BioLegend (1 mentions)
Cd11c apc, by Thermo Fisher Scientific (1 mentions)
Cd11b bv510, by BioLegend (1 mentions)
Igm pe, by BioLegend (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Cls 3, by Worthington (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Cd11b percp cy5, by BioLegend (1 mentions)
Facsymphony s6, by BD (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
70 m cell strainer, by BD (1 mentions)
6 protocols using cd45 bv650
1
Multiparametric Immune Cell Profiling
2
Glial Cell Profiling in Mouse Brain
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Hippocampal homogenates were washed, centrifuged at 500 g for 5 min, and blocked with PBS + 10% rat serum for 1 h, then stained with flow markers (BioLegend and Miltenyi) of anti-mouse Csf1r-Brilliant Violet (BV)605 (135517, BioLegend), CD11b-BV421 (101251, BioLegend), CD45-BV650 (103151, BioLegend), Glast-APC (130–123-555, Miltenyi), and O4-PE (130–117-357, Miltenyi) for 1 h. Washed cells were resuspended in 500 µl PBS and acquired with a Fortessa flow cytometer (BD Bioscience). Data were analyzed by Kaluza v2.1 software (Beckman Coulter). Astrocytes were defined as Glast+ cells, oligodendrocyte precursor cells (OPCs) as O4+ cells, microglia as CD45lowCD11bhi cells. The % of glia among total brain cells and mean fluorescent intensity (MFI) of Csf1r per microglia were measured and total Csf1r protein level was calculated as MFI x microglial No.
3
Quantification of Brain Cell Populations
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Mouse hippocampi were dissected after CO2 euthanization and gently homogenized through 70 μm cell strainers (#352350, BD Bioscience, United States) in ice-cold PBS with 1% FBS. Homogenates were washed and centrifuged at 500 g for 5 min. Isolated cells were blocked with 10% rat serum in ice-cold PBS for 1 h. Brain cells were stained with 0.5 μL anti-mouse VGLUT2-Alexa488 (#MAB5504A4, Millipore, United States), CD11b-BV421 (#101251, BioLegend, United States), CD45-BV650 (#103151, BioLegend, United States), Glast-APC (#130–123-555, Miltenyi, Germany), and O4-PE (#130–117-357, Miltenyi, Germany) in PBS with 1% FBS on ice for 1 h. Corresponding isotype control antibodies (all BioLegend, United States) included rat IgG2a-Alexa488 (##400,525), IgG2b-BV421 (#400639), IgG2b-BV650 (#400651), IgG2b-APC (#400219), and IgM-PE (#401611). Cells were washed, resuspended in 500 mL PBS, and acquired with a Fortessa flow cytometer (BD Bioscience, United States). Data were analyzed by Kaluza v2.1 software (Beckman Coulter, United States). Astrocytes were defined as Glast+ cells, oligodendrocyte precursor cells (OPCs) as O4+ cells, and microglia as CD45lowCD11bhi cells. Cell populations were calculated as % among total brain cells or microglia as previously described (Piirainen et al., 2021 (link); Chithanathan et al., 2022 (link)).
4
Microglia Isolation from Brain Tissue
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Harvested brain tissue were digested in 200 U/mL collagenase III solution (Worthington CLS-3). After 10 min incubation at 37 °C, samples were mechanically dissociated using a P1000 pipette and incubated an additional 10 min. BV2 cells were collected using a sterile cell scraper. Equal volume cold PBS was added, and the samples were filtered through a 70 μm cell strainer. Samples were centrifuged at 200×g for 5 min. After discarding supernatant, samples were resuspended in FACS buffer (PBS containing 2.5% BSA and 2 mM EDTA) containing 1:100 CD16/32 FcR blocking antibody (BD553143) and incubated at 4 °C for 10 min. Next an antibody cocktail containing Zombie Aqua (BioLegend 423,101), CD45-BV650 (BioLegend 1,031,515), CD11b-PerCP/Cy5.5 (BioLegend 101,227), and Tmem119-PE/Cy7 (Invitrogen 25-6119-82) were added at a 1:100 final dilution for all components. The samples were incubated at 4 °C in the dark for an additional 20 min. Samples were washed twice with FACS buffer and data were acquired on a BD LSRII flow cytometer. The CD45intCD11bpos population were sorted on a BD FACSymphony S6.
5
Flow Cytometric Analysis of Brain Cells
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The third cohort of mice was used for flow cytometry analysis. After 24 h of LPS/saline challenge, mice were euthanized by carbon dioxide (CO2) overdose, and the hippocampus and cerebellum were dissected. Dissected brain regions were gently homogenized through 70 µm cell strainers (BD Falcon) in FC buffer (ice-cold, phosphate-buffered saline (PBS) + 1% fetal calf serum). Isolated cells were blocked with 10% rat serum in ice-cold PBS for 1 h. Brain cells were stained with 0.5 μL of anti-mouse CD11b-BV421 (#101251, BioLegend, San Diego, CA, USA), CD45-BV650 (#103151, BioLegend, San Diego, CA, USA), O4-PE (#130-117-357, Miltenyi Biotec, Bergisch Gladbach, Germany), CX3CR1-A488 (#149021, BioLegend San Diego, CA, USA) for 1 h at 4 °C. Cells were washed and resuspended into 0.5 mL PBS. Samples were acquired with a 5-laser LSR Fortessa (BD Biosciences, San Jose, CA, USA) cytometer and analyzed with Kaluza v1.2 software (Beckman Coulter, Indianapolis, IN, USA).
6
Immunophenotyping of PB-MNC Subsets
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PB-MNCs samples from patients were slowly thawed and resuspended in PBS-SVF-EDTA buffer for counting in rst step and resuspended at a concentration of 10 millions of cells per mL. Cells were incubated with an anti-human antibodies cocktail of lineage (α-CD3, α-CD14, α-CD16, α-CD11b, α-CD11c, α-CD19, α-CD56), CD45-BV650 (BioLegend), CD34-PeCy7 (BioLegend), c-Kit (CD117)-PeDazzle594 (BioLegend), CD133-VioBright667 (Miltenyi Biotec) and Zombie NIR (Biolegend) for viability during 30 minutes in the dark. Cells were washed by adding PBS-SVF-EDTA buffer and centrifuged two times.
Before acquisition, cells were xed during 45 minutes in the dark at 4°C. Acquisitions were performed with a three lasers Aurora spectral ow cytometer (Cytek). Unmixing was calculated and applied to samples based on reference single stained PB-MNCs controls. Data were rst analyzed in a supervised way with FlowJo (FlowJo, LLC) to identify immature cells. Down sampling of CD34 + population was done for every sample. Featured on CD45, CD34, CD133 and c-Kit expressions, consecutive dimension reduction was performed by Uniform Manifold Approximation and Projection (UMAP) algorithm (17) and metaclustering was assessed by FlowSOM enabled identi cation of 7 clusters among the CD34 + cells using the cloud-based platform OMIQ (https://www.omiq.ai/).
Before acquisition, cells were xed during 45 minutes in the dark at 4°C. Acquisitions were performed with a three lasers Aurora spectral ow cytometer (Cytek). Unmixing was calculated and applied to samples based on reference single stained PB-MNCs controls. Data were rst analyzed in a supervised way with FlowJo (FlowJo, LLC) to identify immature cells. Down sampling of CD34 + population was done for every sample. Featured on CD45, CD34, CD133 and c-Kit expressions, consecutive dimension reduction was performed by Uniform Manifold Approximation and Projection (UMAP) algorithm (17) and metaclustering was assessed by FlowSOM enabled identi cation of 7 clusters among the CD34 + cells using the cloud-based platform OMIQ (https://www.omiq.ai/).
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