Freshly excised hearts were minced on ice and digested in serum free Iscoves DMEM supplemented with 2mg/mL of Collagenase Type 2 (Worthington, Cat #LS004176) and 0.02mg/mL of Deoxyribonuclease I from bovine pancreas (Sigma, Cat #DN25-100MG) at 37°C for 30 minutes. These cell suspensions were then neutralized with IDMEM containing 10% FBS and filtered through 0.45μm strainer before blocking with 2.4G2 (BD Pharmingen, Cat #553141) for 30 minutes on ice, and staining with Gr-1-APC (Clone RB6-8C5, eBioscience), CD11b-APC-Cy7 (M1/70, BD Pharmingen), Ly 6C-PerCP-Cy5.5 (HK1.4, eBioscience), Ly6G-FITC (1A8, eBioscience), MHC-II PE-Cy5 (M5/114.15.2, eBioscience), MerTK-PE (DS5MMER, eBIoscience), F4/80-PE-Cy5 (BM8, eBioscience) and CD64-Biotin (X54-5/7.1, Biolegend and used in conjunction with Streptavidin PE-Cy7, eBioscience) antibodies for 30 minutes on ice protected from light. Cells were washed with PBS prior to flow cytometry. The samples were then analyzed on BD LSR-II (UAB Flow Cytometry Core Facility). Neutrophils were identified as Gr-1+CD11b+Ly-6G+F4/80-MHC-II-. Cardiac macrophages were first gated on F4/80+CD11b+ cells and further gated for expression of MerTK and CD64. Flow cytometry data were analyzed using FlowJo software. Percent neutrophils and macrophages were determined as (Percent live gated cells from the heart) x (percent positive).
F4 80 pe cy5 bm8
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The F4/80-PE-Cy5 (BM8) is a fluorescence-conjugated antibody that can be used to detect and analyze the F4/80 antigen, which is commonly expressed on the surface of macrophages. This product is suitable for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b apc cy7, by BD (1 mentions)
Gr 1 apc clone rb6 8c5, by Thermo Fisher Scientific (1 mentions)
Mhc 2 pe cy5, by Thermo Fisher Scientific (1 mentions)
Streptavidin pe cy7, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Streptavidin bv650, by BioLegend (1 mentions)
Cyan adp 9, by Beckman Coulter (1 mentions)
Gr 1 fitc rb6 8c5, by BD (1 mentions)
Easysep cd11b positive selection kit, by STEMCELL (1 mentions)
Kaluza, by Beckman Coulter (1 mentions)
Moflo astrio, by Beckman Coulter (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
3 protocols using f4 80 pe cy5 bm8
1
Isolation and Identification of Cardiac Immune Cells
2
Multicolor Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated mononuclear cells from PB, spleen and dLNs were surface stained with antibodies directed against mouse CD45PerCP (30F11.1), Ly6C‐FITC (1G7.G10), and Ly6G‐PerCPVio770 (1A8) from Miltenyi Biotech (Bergisch Gladbach, Germany), F4/80‐PECy5 (BM8) from eBiosciences (San Diego, CA), streptavidin‐BV650 from Biolegend (San Diego, CA), and CD11b‐biotin (M1/70), Gr1‐biotin (RB6‐8C5), Ly6G‐BV421 (1A8), and isotype‐matched control IgG from Becton Dickinson Pharmingen (Franklin Lakes, NJ). Cells were collected on a BD LSR Fortessa flow cytometer (Becton Dickinson). At least 10,000 events were acquired. Data were analyzed using FlowJo software (Ashland, OR).
3
VSV-Mediated Cytokine Induction in EMT6 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EMT6 cells were cotreated with 0.1 MOI of VSVΔ51-GFP and 5 μM LCL161 for 20 hr. Cells were trypsinized, permeabilized with the CytoFix/CytoPerm kit (BD Biosciences) and stained with APC-TNFα (MP6-XT22, BD Biosciences). Cells were analyzed on a Cyan ADP 9 flow cytometer (Beckman Coulter) and data was analyzed with FlowJo (Tree Star).
Splenocytes were enriched for CD11b using the EasySep CD11b positive selection kit (StemCell Technologies). CD49+ cells were enriched using the EasySep CD49b positive selection kit (StemCell Technologies) from the CD11b− fraction. CD11b+ cells were stained with F4/80-PE-Cy5 (BM8, eBioscience) and Gr1-FITC (RB6-8C5, BD Biosciences) and further sorted with MoFlo Astrios (Beckman Coulter). Flow cytometry data was analyzed using Kaluza (Beckman Coulter). Isolated cells were infected with VSVΔ51 for 24 hr and clarified cell culture supernatants was applied to EMT6 cells for 24 hr in the presence of 5 μM LCL161. An n = 3 of biological replicates was used to determine statistical measures (mean, standard deviation).
Splenocytes were enriched for CD11b using the EasySep CD11b positive selection kit (StemCell Technologies). CD49+ cells were enriched using the EasySep CD49b positive selection kit (StemCell Technologies) from the CD11b− fraction. CD11b+ cells were stained with F4/80-PE-Cy5 (BM8, eBioscience) and Gr1-FITC (RB6-8C5, BD Biosciences) and further sorted with MoFlo Astrios (Beckman Coulter). Flow cytometry data was analyzed using Kaluza (Beckman Coulter). Isolated cells were infected with VSVΔ51 for 24 hr and clarified cell culture supernatants was applied to EMT6 cells for 24 hr in the presence of 5 μM LCL161. An n = 3 of biological replicates was used to determine statistical measures (mean, standard deviation).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!