Each spleen was placed in FACS buffer (0.5% BSA, 2mM EDTA in PBS) and processed into single cell suspension using a gentleMACS™ Dissociator. Total spleen cell counts were obtained by counting of a fraction of the diluted cells by flow cytometry. Red blood cells (RBC) were lysed using BD Pharm Lyse lysis buffer (BD Biosciences) prior to the addition of mouse FcR Blocking Reagent (Miltenyi Biotec). Cells were stained with the following fluorochrome-conjugated antibodies: CD3ε-VioBlue (clone: 17A2, Miltenyi Biotec), APC-Cy™7 Rat Anti-Mouse CD4 (clone: GK1.5 (RUO), BD Biosciences), PE Rat Anti-Mouse CD8a (clone: 53–6.7 (RUO), BD Biosciences), PerCP/Cy5.5 anti-mouse TCR β chain (clone: H57–597, BioLegend), FITC anti-mouse CD19 (clone: 6D5, BioLegend), CD45R (B220)-APC, (clone: RA3–6B2, Miltenyi Biotec), and PE/Cy7 anti-mouse CD11c (clone: N418, BioLegend). Stained cells were then analyzed by flow cytometry using a MACSQuant® Analyzer 10 (Miltenyi Biotec) and with WinList Version 9 software (Verity Software House). The following cell types were gated based on FSC versus SSC and positive expression of their respective markers: B cells (TCRβ-CD19+ cells), total T cells (TCRβ+ cells), CD4+ T cells (TCRβ+CD4+ cells), CD8+ T cells (TCRβ+CD8+ cells), and double-negative (DN) T cells (TCRβ+CD4-CD8- cells). An example of the gating strategy is shown in Supplementary Figure 2 .
Fitc anti mouse cd19
Manufactured by BioLegend
Sourced in United States
FITC anti-mouse CD19 is a fluorescently labeled monoclonal antibody that binds to the CD19 antigen expressed on mouse B cells. It can be used to identify and quantify B cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Apc cy7 anti mouse cd11c, by BioLegend (2 mentions)
Fitc anti mouse cd3ε, by BioLegend (2 mentions)
Pe cy7 anti mouse cd45, by BioLegend (2 mentions)
Pe rat anti mouse cd8a, by BD (1 mentions)
Pharm lyse lysis buffer, by BD (1 mentions)
Apc cy 7 rat anti mouse cd4, by BD (1 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Apc anti mouse ly6g, by BioLegend (1 mentions)
Lsr 2, by BD (1 mentions)
Pacific blue anti mouse cd11b, by BioLegend (1 mentions)
Pe cy7 anti mouse f4 80 clone bm8, by BioLegend (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti icos, by Abcam (1 mentions)
Anti cd40, by Abcam (1 mentions)
Anti cd40l, by Abcam (1 mentions)
Anti icosl, by Abcam (1 mentions)
Anti cd4, by Santa Cruz Biotechnology (1 mentions)
Anti mouse cd16 32, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
9 protocols using fitc anti mouse cd19
1
Multiparametric Flow Cytometry of Murine Splenocytes
2
Quantification of Lung Cell Types
3
Comprehensive Immunophenotyping of Murine Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following Abs and reagents were used in this study: FITC anti-mouse CD3ε (#100306), FITC anti-mouse CD19 (#152404), APC anti-mouse CD21/CD35 (#123412), PerCP/Cyanine5.5 anti-mouse CD23 (#101618), and Brilliant Violet 421 anti-mouse IgD (#405725) (all from BioLegend, CA, USA); PE anti-mouse CD45 (#12-0451-82) (Invitrogen, CA, USA); anti-TCR α/β (#sc-19600), anti-CD4 (#sc-19641), and anti-GAPDH (#sc-365062) (Santa Cruz Biotechnology, TX, USA); anti-MHC Class II (#ab180779), anti-ICOS (#ab175401), anti-ICOSL (#ab138354), anti-CD40 (#ab188181), and anti-CD40L (#ab2391) (Abcam, Cambridge, UK); and FITC anti-mouse CD169 (MOMA-1, #MCA947F) (Bio-Rad, CA, USA).
4
Multiparameter Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood was drawn via cardiac puncture and incubated with ammonium-chloride-potassium lysing buffer for red blood cell lysis. Blood leukocytes were subsequently blocked with anti-mouse CD16/32 (14–0161–81; eBioscience) and stained with fluorophore-conjugated antibodies A700 anti-mouse CD45 (103127; BioLegend), PE anti-mouse CD11b (101208; BioLegend), APC-Cy7 anti-mouse CD11c (117323; BioLegend), PE-Cy7 anti-mouse CD4 (100421; BioLegend), APC anti-mouse CD8 (100711; BioLegend) and FITC anti-mouse CD19 (115505; BioLegend) as well as propidium iodide (PI; Invitrogen) for live/dead discrimination. Data were acquired on a BD FACSAria II and analyzed using the FlowJo software to assess peripheral cell counts. Gating was performed following routinely used protocols, using FSC/SSC to exclude dead cells, cell debris and doublets and propidium iodide to identify dead cells. At least 250,000 events were captured by the cytometer and 30,000 single cells analyzed per group. Cells were gated on CD45+PI− live singlets. Dead cells were excluded as propidium iodide (PI)-positive. Populations of live singlets were then gated was percentages of CD45 + PI- cells.
5
Flow Cytometry Analysis of Biotin+ B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry data analyses were performed using FlowJo software (10.6.2). tSNE analysis was performed using FCS Express 7 from De Novo software. Cells were suspended in T cell medium supplemented with anti-mouse CD16/32 to block Fc receptors before staining with fluorescent antibodies against cell surface epitopes. Samples were stained using the following antibodies: zombie, FITC anti-mouse CD19, PB anti-mouse CD45.1, APC streptavidin, and anti-mouse CD16/32 TruStain FcXPLUS purchased from BioLegend. First, dead cells were excluded through zombie, then splenocytes were distinguished through anti-mouse CD45.1, B cells were isolated through anti-mouse CD19, and last, Biotin+ B cells and Biotin− B cells were sorted through streptavidin. The background was defined as the signal produced on B cells by incubating with iDCs without membrane-anchored sFT.
6
Murine Allergic Inflammation Assay
7
Lymphocyte Immunophenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lymphocytes were isolated as mentioned in Section 4.5 . After counting and washing with fresh sterile PBS (with 5% FBS, 500× g, 5 min) three times, the pelleted cells were re-suspended to 1 × 107 cell/mL in PBS. The cell samples were divided into two tubes, 100 μL each, and antigens were added: PE/Cy7-anti-mouse CD3 (0.5 μg, the same below), FITC-anti-mouse CD4, PE-anti-mouse CD8a, and FITC-anti-mouse CD19, respectively (all Biolegend, San Diego, CA, USA) [41 ]. After mixing and incubation in dark at 4 °C for 30 min, the cells were washed with 900 μL cold-PBS twice, centrifuged (500× g, 5 min), and re-suspended with 300 μL PBS. The stained cells were analyzed with BD FACS Verse flow cytometer with FACSuite software (Becton Dickinson). Lymphocyte populations were determined as the percentages of T cells (CD3+, CD19−) and B cells (CD3−, CD19+) among leukocytes. Subpopulations of helper T cells and cytotoxic T cells are presented as the percentage of CD4+CD8− and CD4−CD8+ cells among CD3+-expressing cells.
8
Lymphocyte Profiling via Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the euthanasia, 200 µL of blood was collected into 1.5 mL tubes (Eppendorf, Germany) containing 100 µL of 0.5 M EDTA. For flow cytometry, erythrocytes were disrupted using RBC Lysing Buffer (0.15 M ammonium chloride, 10 mM sodium bicarbonate, and 0.1 mM EDTA). After the lysis of erythrocytes, the remaining cells were washed twice.
Profiling of the peripheral blood lymphocyte population was performed on a BD FACSCanto™ II flow cytometer. Staining was performed according to the manufacturer’s protocol with an appropriate combination of fluorescent monoclonal antibodies: anti-mouse CD45-PE/Cy7, anti-mouse CD19-FITC, anti-mouse CD4, anti-mouse CD8-PE/Cy7, and anti-mouse CD3-PE (all from BioLegend, San Diego, CA, USA).
Profiling of the peripheral blood lymphocyte population was performed on a BD FACSCanto™ II flow cytometer. Staining was performed according to the manufacturer’s protocol with an appropriate combination of fluorescent monoclonal antibodies: anti-mouse CD45-PE/Cy7, anti-mouse CD19-FITC, anti-mouse CD4, anti-mouse CD8-PE/Cy7, and anti-mouse CD3-PE (all from BioLegend, San Diego, CA, USA).
9
Antibody Sources for Murine Immunoassays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in ELISAs for IgG, IgG subclasses, and IgE were purchased as follows: goat anti-mouse IgG-horseradish peroxidase (HRP) was purchased from Sigma-Aldrich (St Louis, Mo), and the following antibodies were all purchased from Abcam (Cambridge, United Kingdom): goat anti-mouse IgE-HRP, goat anti-mouse IgG 1 -HRP, goat anti-mouse IgG 2a -HRP, goat anti-mouse IgG 2b -HRP, and goat anti-mouse IgG 3 . Antibodies for flow cytometric analyses were purchased from BioLegend (San Diego, Calif), as follows: anti-mouse CD45 in fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin, and peridinin-chlorophyllprotein complex/Cy5.5 and anti-mouse CD19-FITC, anti-mouse CD124-PE, and mouse isotype controls in FITC, PE, allophycocyanin, and peridinin-chlorophyll-protein complex/Cy5.5. Antibody against serotype 19 pneumococcal strains (Rb anti-S pneumoniae serotype 19) was provided by Dr Moon Nahm and Dr Brady Spencer (University of Alabama at Birmingham, Birmingham, Ala) for this work. Total IgE levels were examined by using a specific kit for Mouse IgE (BioLegend), according to the manufacturer's instructions. M pneumoniae P1-adhesin carboxy terminus (recombinant P1 [rP1]) peptide was purchased from ProSpec Biosciences (Ness-Ziona, Israel). Grade V ovalbumin (OVA) was purchased from Sigma-Aldrich for immunization, airway challenge, and enzyme immunoassay.
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!