Blood (50 µL) from tail vein was collected using heparin-coated capillary tubes, spun down, and erythrocytes were lysed with ACK buffer for 5 min in ice followed by a washed with 1% BSA in PBS. Splenocytes were isolated as previously described (19 (link)). Cells were blocked with anti-mouse Fc block (BD Biosciences) and surface stained with antibodies against human CD3 (#HIT3a), CD4 (#SK3), CD8 (#RPA-T8), CD69 (#L78), CD62L (#DREG-56), and CCR7/CD197 (#150503) from BD Biosciences as described (18 (link)–20 (link)). To evaluate the frequency of human CD4+FOXP3+ regulatory T cells in spleens of DRAGA mice, cells were first surface stained with human CD3, CD4 antibodies, and then intracellularly stained with a antibody against human FOXP3 (#236A/E7, Thermo Fisher Scientific) following the manufacturer’s instructions. Cells were analyzed in the gated mononuclear FSC/SSC as described previously (19 (link)).
Anti mouse fc block
Manufactured by BD
The Anti-mouse Fc Block is a reagent designed for use in flow cytometry and immunofluorescence applications. It is used to prevent non-specific binding of antibodies to Fc receptors on the surface of mouse cells, thereby reducing background staining and improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3 sorter, by BD (4 mentions)
Perm wash buffer, by BD (4 mentions)
7 aad, by BD (3 mentions)
Anti mouse fitc conjugated anti cd45 clone 30 f11, by BD (3 mentions)
Collagenase type 1, by Merck Group (3 mentions)
Dnase, by Merck Group (3 mentions)
Cytomics fc500, by Beckman Coulter (3 mentions)
Cytofix cytoperm, by BD (2 mentions)
Normal rat serum, by Jackson ImmunoResearch (2 mentions)
Hit3a, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Totalseq c antibodies, by BioLegend (1 mentions)
Automacs, by Miltenyi Biotec (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Anti cd45 microbeads, by Miltenyi Biotec (1 mentions)
Rat serum, by Jackson ImmunoResearch (1 mentions)
Cd11b bv421, by BioLegend (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
Sirpαpe, by BioLegend (1 mentions)
Fixable viability dye e506, by Thermo Fisher Scientific (1 mentions)
11 protocols using anti mouse fc block
1
Immunophenotyping of Human T Cells in DRAGA Mice
2
Immunophenotyping of Leukemic Mouse Cells
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Bone marrow isolated from leukemic mice was blocked with anti-mouse Fc Block (BD Biosciences, dilution 1:50) for 10 min at 4 °C. Surface staining with anti-CD45-APC/Cy7 (dilution 1:100) and DPP4-PE (dilution 1:20) was performed at 4 °C for 30 min. Following this, the samples were washed in Medium-199 supplemented with 2% FBS and then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4 °C. The fixed cells were thereafter washed with 1× Perm/Wash buffer (BD Biosciences) and resuspended in Perm/Wash buffer containing anti-Ki67-AF647 at a 1:10 dilution for a 30 min incubation. The samples were washed one more time with 1× Perm/Wash buffer (BD Biosciences) and then incubated in 1× Perm/Wash buffer (BD Biosciences) with 2 μg ml−1 DAPI (Biolegend) for 10 min. Finally, the samples were spun down to remove the DAPI-containing buffer and resuspended in Medium-199 supplemented with 2% FBS for analysis. Flow cytometry was performed on a BD FACS Aria III sorter (BD Biosciences) and all data were analyzed using FlowJo (Treestar).
3
Bone Marrow Immunophenotyping by Flow Cytometry
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Whole bone marrow and micropipette samples from calvarium and tibia were blocked with anti-mouse Fc Block (BD Biosciences, dilution 1:50) for 10 min at 4 °C. The cells were thereafter stained with blood cell lineage cocktail (Supplementary Table 6 ) for 30 min at 4 °C. For detection of dead cells 7AAD (BD Biosciences, 0.25 µg) was added to the sample prior to analysis. Flow cytometry was performed on a BD FACS Aria III sorter (BD Biosciences) and all data were analyzed using FlowJo (Treestar).
4
Immunophenotyping Leukemic Bone Marrow
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Leukemic bone marrow was blocked using anti-mouse Fc Block (BD Biosciences, dilution 1:50) for 10 min at 4 °C in Medium-199 supplemented with 2% FBS. Surface staining was thereafter performed with a leukemia cluster cocktail (Supplementary Table 6 ) for 30 min at 4 °C. The cells were then washed and resuspended in Medium-199 supplemented with 2% FBS with 0.25 µg 7AAD (BD Biosciences). Flow cytometry was performed on a BD FACS Aria III sorter (BD Biosciences) and all data were analyzed using FlowJo (Treestar).
5
Single-Cell Isolation from Adipose Tissue
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Mice were euthanized by isoflurane overdose and cervical dislocation and perfused with 20 ml PBS through the left ventricle. eAT pads were collected, minced, and digested in 6 ml of 2-mg/mL type II collagenase (Worthington # LS004177) for 30 min at 37 °C. Digested eAT was then vortexed, filtered through 100 μm filters, lysed with ACK buffer, and filtered through 35 μm filters as previously described81 .
The AT stromal vascular fraction was prepped with anti-mouse Fc Block (BD Biosciences) at 1:200. Cells from each mouse were labeled with unique hashtag antibodies (1:200) (Biolegend TotalSeq-C) and anti-CD45 microbeads (10 μL/ sample) (Miltenyi # 130-052-301). Biological replicates were pooled and sorted on a Miltenyi AutoMACs using the “possel_s” option. CD45+ cells were then labeled for CITE-sequencing using TotalSeq-C antibodies (Biolegend) for cell surface markers to identify major cell types. All immunolabeling was completed at a 1:200 dilution for 20 min at 4 °C in the dark. More information regarding specific samples and antibody manufacturer/catalog numbers can be found in Supplementary Table1 . Cells were stained with 0.25 µg/mL DAPI for FACS sorting and DAPI− viable cells were collected for downstream processing and sequencing.
The AT stromal vascular fraction was prepped with anti-mouse Fc Block (BD Biosciences) at 1:200. Cells from each mouse were labeled with unique hashtag antibodies (1:200) (Biolegend TotalSeq-C) and anti-CD45 microbeads (10 μL/ sample) (Miltenyi # 130-052-301). Biological replicates were pooled and sorted on a Miltenyi AutoMACs using the “possel_s” option. CD45+ cells were then labeled for CITE-sequencing using TotalSeq-C antibodies (Biolegend) for cell surface markers to identify major cell types. All immunolabeling was completed at a 1:200 dilution for 20 min at 4 °C in the dark. More information regarding specific samples and antibody manufacturer/catalog numbers can be found in Supplementary Table
6
Isolation and Quantification of Murine Corneal Leukocytes
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Corneas were dissected from mouse eyes at 4 days post infection (dpi) and cut into small (1–2 mm diameter) fragments for subsequent digestion with 1 mg/ml collagenase type I and 0.5 mg/ml DNase (Sigma Chemical, St. Louis, MO)18 (link). Single cell suspensions were washed twice (300 × g, 5 min/wash) in PBS and then incubated on ice for 15 min with 2 µl anti-mouse Fc block (BD Pharmingen, San Diego, CA) in a total volume of 100 µl PBS-1% BSA. Following incubation, cells were centrifuged (300 × g, 5 min) and resuspended in 5% normal rat serum (Jackson Immuno Research, West Grove, PA) for an additional 15 min on ice. Cells were then labeled with 4 µl anti-mouse FITC-conjugated anti-CD45 (clone 30-F11, BD Pharmingen), and incubated in the dark on ice for 30 min. Following incubation, the cells were washed 3 times with PBS-1% BSA (300 × g, 5 min/wash) and resuspended in PBS-1% BSA and flow cytometry performed using a Cytomics FC500 (Beckman Coulter, Brea, CA) for CD45+ events, representing the numbers of fluorescent events identified in infected and control corneas and roughly corresponding to the number of leukocytes per cornea.
7
Isolation and Quantification of Murine Corneal Leukocytes
8
Flow Cytometry of Corneal Leukocytes
9
Microglia Isolation and Characterization
10
Leukemic Bone Marrow Immunophenotyping
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Leukemic bone marrow was blocked using anti-mouse Fc Block (BD Biosciences, dilution 1:50) for 10 min at 4 °C in Medium-199 supplemented with 2% FBS. Surface staining was thereafter performed with CD45-APC/Cy7 (BD Biosciences, dilution 1:100) and DPP4-PE (BioLegend, dilution 1:20) for 30 min at 4 °C. The cells were then washed and resuspended in Medium-199 supplemented with 2% FBS with 0.25 µg 7AAD (BD Biosciences). Flow cytometry was performed on a BD FACS Aria III sorter (BD Biosciences) and all data were analyzed using FlowJo (Treestar). Extended Data Fig. 8c shows the gating strategy used to distinguish DPP4high, DPP4int and DPP4neg cells. Note that DPP4-positive cells were defined as DPP4high and DPP4int.
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