A 4 × 4 mm cortical tissue was microdissected in ice-cold L15 dissecting media (ThermoFisher, Waltham, MA) and subjected to papain neural dissociation (Miltenyi Biotec, Auburn, CA) per manufacturer instructions. Blood samples were prepared from 1 ml of cardiac puncture collected in EDTA, subjected to ACK lysing buffer, resuspended in 100ul PBS per sample and placed in a 96-well plate. Cells were incubated at 1/500 in Zombie aqua (BioLegend, San Diego, CA) for 20 m at room temperature, washed and centrifuged at 300g at 4 °C for 5 min. Cells were resuspended in PBS/ 1% FBS, washed then incubated with 2% FC blocker in 100ul of PBS/1% FBS/2 mM EDTA on ice for 10 min, followed by 20 m primary antibody incubation (APC-CD45; Alexa Fluor 700-CD11b; PE/Cy7- Ly6G; BV-421-Ly6C; Alexa Fluor 488-CD115; BV-650-CCR2; BioLegend, San Diego, CA). Unstained and single-color controls for each antibody target were included to set limits in flow analysis. BD FACSAria™ II Flow Cytometer was used to collect ~ 50k cells per sample and data analyzed using FlowJo v10 (BD, Ashland, Oregon).
Cd11b alexa fluor 700
Manufactured by BioLegend
Sourced in United States
CD11b Alexa Fluor 700 is a fluorescently-labeled monoclonal antibody that specifically binds to the CD11b cell surface antigen. CD11b is a component of the integrin Mac-1 (CD11b/CD18), which is expressed on the surface of various immune cells, including monocytes, macrophages, and granulocytes. The Alexa Fluor 700 fluorescent dye is conjugated to the antibody, allowing for detection and analysis of CD11b-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
L15 dissecting media, by Thermo Fisher Scientific (1 mentions)
Facsaria 2 flow cytometer, by BD (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Cd45 apc, by BioLegend (1 mentions)
Ly6g pe cy7, by BioLegend (1 mentions)
Percp cyanine5.5 ly6g, by BioLegend (1 mentions)
Live dead fixable far red dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Percoll, by Merck Group (1 mentions)
Mhc 2 efluor450, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc gk1, by Thermo Fisher Scientific (1 mentions)
Cd8a percp cyanine5, by Thermo Fisher Scientific (1 mentions)
Cd25 apc pc61, by Thermo Fisher Scientific (1 mentions)
Foxp3 pe nrrf 30, by Thermo Fisher Scientific (1 mentions)
Fortessa x20, by BD (1 mentions)
Cd14 fitc, by Thermo Fisher Scientific (1 mentions)
F4 80 pe, by BioLegend (1 mentions)
Intracellular fixation and permeabilization buffer kit, by Thermo Fisher Scientific (1 mentions)
Anti cd45 pe cy5 clone 30 f11, by BioLegend (1 mentions)
Anti ly 6g pe cy7 clone 1a8, by BioLegend (1 mentions)
6 protocols using cd11b alexa fluor 700
1
Multicolor Flow Cytometry Profiling
2
Liver Lymphocyte Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lymphocytes isolated from livers by percoll (Sigma, St. Louis, MO, USA) were washed three times in phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS; FACS Buffer). For detecting dead cells, harvested cells were stained via a LIVE/DEAD Fixable Far-Red-Dead Cell Stain Kit (1:1000) using either 633 or 635 nm excitation (Cat#L34973, Thermo Fisher Scientific). For Brilliant Violet 510-CD45 (Cat#103137, BioLegend) (1:400), Alexa Fluor 700-CD11b (Cat#101222, BioLegend) (1:400), PerCP/Cyanine5.5-Ly6G (Cat#127616, BioLegend) (1:600), and PE/Cyanine7-F4/80 (Cat#123114, BioLegend) (1:400) staining, harvested cells were washed and incubated in PBS containing 1% FBS containing fluorochrome-conjugated antibodies in a U-bottom 96-well plate. The frequency of the different cell population was measured via flow cytometry (BD LSRFortessa, BD Biosciences) and analyzed by FlowJo_v10 (BD, Ashland, OR, USA). Antibodies used in this study for FC were listed in Supplementary Materials
3
Multiparametric Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell suspensions were stained with combinations of following monoclonal fluorescently conjugated antibodies: TCRβ eFluor® 450 (H57-597, eBioscience, San Diego, CA, USA), CD4 FITC (GK1.5, eBioscience), CD8a PerCP-Cyanine5.5 (53–6.7, eBioscience), CD25 APC (PC61.5, eBioscience), FoxP3 PE (NRRF-30, eBioscience), CD45R/B220 APC-Cyanine7 (RA3-6B2, Biolegend, San Diego, CA, USA), IgD Brilliant violet 650™ (11-26c.2a, Biolegend), CD11b Alexa Fluor® 700 (M1/70, Biolegend), F4/80 PE (BM8, Biolegend), PD-1 PE-eFluor™ 610 (J43, eBioscience), CTLA-4 PerCP-Cyanine5.5 (UC10-4B9, Biolegend), IL-10 PerCP-Cyanine5.5 (JES5-16E3, Biolegend), LAP PerCP-Cyanine5.5 (TWT-16B4, Biolegend), CD14 FITC (Sa2-8, eBioscience), CD206 APC (MR6F3, eBioscience), MHC-II e Fluor® 450 (M5/114.15.2, eBioscience). To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with intracellular fixation and permeabilization buffer kit (eBioscience) according to the manufacturer’s recommendation. For intracellular IL-10 staining, cells were incubated with PMA (50 ng/ml) and ionomycin (1000 ng/ml) for 2 h, then incubated with Brefeldin A (10.6 μM) and Monensin (2 μM) for 3 h. Data were acquired using BD Fortessa X20 (BD Bioscences, San Diego, CA, USA) and analyzed with FlowJo 7.6 software.
4
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Abs used for cell-surface staining included: anti-CD3 Pacific Blue (clone 17A2), anti-CD8 PE Dazzle 594 (clone 53.6.7), anti-CD45 PE-Cy5 (clone 30-F11), anti-Ly-6G PE-Cy7 (clone 1A8), anti-Ly-6C APC-Cy7 (clone AL-21), anti-PD-L1 PE (clone 10F.9G2), anti-PD-1 APC (clone 29F-1A12), CD11b Alexa Fluor 700 (clone M1/70), anti-CD4 Brilliant Violet 570 (clone RM4-5), anti-CD44 PE-Cy7 (clone IM7), CD25 APC (clone 3C7) (Biolegend, San Diego, CA, USA), and CD11c FITC (clone HL3) (BD Biosciences, San José, CA, USA). The abs for intracellular staining were anti-FoxP3 Alexa Fluor 488 (clone MF23) (BD Biosciences) and anti-CTLA-4 PE (clone UC10-4F10-11) (Biolegend). A LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies, Thermo Scientific, Eugene, OR, USA) was used for dead cell exclusion. The abs used for intracellular cytokines evaluation were anti-IFNγ Alexa Fluor 700 (clone XMG1.2), TNFα PE-Cy7 (clone MP6-XT22), IL-2 FITC (clone JES6-5H4) (BD Biosciences), anti-perforin (clone S16009A), and anti-granzyme B (QA16A02) (Biolegend).
5
Murine Lung Infection with B. cenocepacia
6
Multiparametric Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Digested cells were incubated on ice with FcR block for 10 min followed by viability staining with Fixable Viability Dye eFluor506 (eBioscience, Vienna, Austria) for 30 min on ice. Unless indicated otherwise, antibodies were purchased from eBioscience, and utilized with appropriate isotype controls: CD4-Brilliant Violet 785 (BioLegend, Lucerne, Switzerland), CD69-APC-eFluor 780, DO11.10-PE, CD11c-Brilliant Violet 785 (BioLegend), CD11b-Alexa Fluor 700, MHCII-Brilliant Violet 711 (BioLegend), CD86-Brilliant Violet 605 (BioLegend), CD80-Brilliant Violet 605, CD40-PerCP-eFluor 710, CD8α-PE-eFluor610, PD-L1-PE-Cy7 (BioLegend), PD-L2-FITC, and ICOS-L-PE. Intracellular cytokine staining was performed by using 20 μg/ml Brefeldin A (eBioscience) to stop protein transport. Subsequently, surface marker-stained cells were fixed in a 1% formalin solution followed by intracellular staining with the following antibodies with appropriate isotype control diluted in permeabilization buffer [PBS (Sigma) + 0.1% saponin (Sigma) + 10% FCS (Gibco; Thermo Fisher Scientific, Waltham, MA, USA)]: FoxP3-AlexaFluor 700, IL-4-PE-Cy7, IL-17A-Per-CP-Cy5.5, IFNγ-eFluor450, and IL-9-eFluor660. Acquisition was performed by using a SORP LSRII (BD Biosciences) flow cytometer and data were analyzed by using FlowJo X software (Tree Star, Ashland, OR, USA) and FlowJo9 for T cell proliferation.
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!