Inguinal dLNs and spleens were prepared as described42 (link). Cells were transferred to 96 well V-bottom plates (Thermo Scientific, UK) and centrifuged (300 × g, 5 min). Supernatants were then discarded and cells were resuspended with addition of Fc Receptor Block (1:50) (eBioscience, UK) for 15 min. Following a wash in FACS buffer, cells were stained with anti-GL7 AlexaFluor 647 (eBioscience, UK) at 1:100 dilution, anti-CD95 PE (eBioscience, UK) at 1:100 dilution and anti-B220 PeCy7 (eBioscience, UK) at 1:200 for 30 min at RT in the dark. After this incubation, cells were washed twice and resuspended in FACS buffer and data were acquired on a LSRII (BD Bioscience, UK) and analyzed by FlowJo (TreeStar Inc, USA).
Fc receptor block
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Fc receptor block is a laboratory reagent designed to minimize non-specific binding of antibodies to Fc receptors. It functions by occupying Fc receptors, preventing undesirable interactions that could interfere with experimental results.
Automatically generated - may contain errors
Lab products found in correlation
V bottom plate, by Thermo Fisher Scientific (1 mentions)
Anti b220 pecy7, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Fortessa, by BD (1 mentions)
Dnase 1, by Roche (1 mentions)
Dispase 2, by Roche (1 mentions)
F4 80, by BioLegend (1 mentions)
Collagenase d, by Roche (1 mentions)
Anti cd11b apc, by Thermo Fisher Scientific (1 mentions)
Anti ly6g pe clone 1a8, by BD (1 mentions)
Zombie violet fixable viability kit, by BioLegend (1 mentions)
Collagenase a, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Cyan adp cytometer, by Beckman Coulter (1 mentions)
Pe conjugated anti cd86, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti cd11c, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd40, by Thermo Fisher Scientific (1 mentions)
4 protocols using fc receptor block
1
Multi-parameter Analysis of Germinal Center B Cells
2
Melanoma Tumor Microenvironment Dissection
3
Quantification of Peritoneal Neutrophils and Dendritic Cells by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantitate peritoneal neutrophils by flow cytometry, cells from 1 ml of lavage were washed with 1% BSA in PBS, incubated with normal serums and Fc receptor block (eBioscience), and stained with anti-Ly6G-PE (clone 1A8) (BD Biosciences) and anti–CD11b-APC (eBioscience) antibodies. To phenotype DCs, skin tissues or popliteal lymph nodes were minced and incubated for 30 min in collagenase A (100 U/ml; Sigma-Aldrich) in Hanks’ balanced salt solution containing FBS and DNase I. Single-cell suspensions were prepared by straining the digested tissues through a 70-μm cell straining filter (BD Biosciences). Cells were then washed with 1% BSA in PBS supplemented with FBS and 10 mM EDTA, blocked, and stained with fluorochrome-tagged antibodies to CD45, CD64, IA/IE, CD11b, CD11c, CD207, CD301b, and CD103 (BioLegend). Dead cells were detected using a Zombie Violet fixable viability kit (BioLegend). Cells stained with a single antibody and isotype controls were used as needed. To detect neutrophils in circulation, peripheral blood was collected in heparin tubes, stained with antibodies, and red blood cells were lysed using FACS lysing solution (BD Biosciences). Upon fixation with PFA, stained cells were analyzed by a BD FACSCalibur or FACSCanto II flow cytometer and FlowJo software version 10.1. Total cells in the peritoneal lavages or lymph nodes were counted using a hemocytometer.
4
Phenotypic Analysis of Bone Marrow Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow-derived dendritic cells were stained in FACS buffer (PBS containing 0.1% bovine serum albumin and 5 mM EDTA) using the following monoclonal antibodies in the indicated concentration for cell surface markers (all obtained from eBioscience): PE-conjugated anti-CD86 (2 μg/ml), FITC conjugated anti-CD-40 (5 μg/ml), PE-conjugated anti-CD11c (1 μg/ml), and APC-conjugated MHC-II (0.28 μg/ml). Cells were first incubated with Fc receptor block, 5 μg/ml (eBioscience) for 10 min to block any non-specific binding and subsequent staining steps were performed for 20 min at 4°C, followed by washing with FACS buffer. An isotype-matched control staining was done for each antibody to determine a specific background staining. Cells were acquired using a Cyan-ADP cytometer (Beckman Coulter, Woerden, The Netherlands) and analyzed with FlowJo software (FlowJo LLC, Ashland, OR, USA).
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!