BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through a 40 μm nylon mesh and then sorted by FACS. Anti-CD45 (BioLegend, 157607, 1:200 dilution) and anti-CD3 (BioLegend, 100209, 1:200 dilution) antibodies were used for the isolation of T cells. Anti-CD45 and anti-CD19 (BioLegend, 152407, 1:200 dilution) antibodi were used for isolation of B cells. Anti-CD45 and anti-NK-1.1 (BioLegend, 108703, 1:200 dilution) antibody were used for isolation of NK cells. Anti-CD45 and anti-F4/80 (BioLegend, 123105, 1:200 dilution) antibodies were used for the isolation of macrophages. Anti-CD45 and anti-Ly6G (BioLegend, 127627, 1:200 dilution) antibodies were used for the isolation of neutrophils. Anti-EpCAM (BioLegend, 324215, 1:200 dilution) and anti-SP-C (abclonal, A1835, 1:500 dilution) antibodies were used for the isolation of alveolar epithelial cells. anti-CD31 (BioLegend, 102409, 1:200 dilution) antibody was used for the isolation of endothelial cells. Anti-S100A4 (abcam, ab197896, 1:500 dilution) antibody was used for the isolation of fibroblasts.
Anti cd45
Manufactured by BioLegend
Sourced in United States, Germany
Anti-CD45 is a lab reagent used for the identification and analysis of leukocytes, the white blood cells that are essential for the immune response. It is a monoclonal antibody that specifically binds to the CD45 protein, which is expressed on the surface of all leukocytes. This reagent can be used in various immunological techniques, such as flow cytometry, to detect and quantify different types of white blood cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b, by BioLegend (75 mentions)
Anti cd3, by BioLegend (61 mentions)
Anti cd4, by BioLegend (57 mentions)
Anti cd8, by BioLegend (49 mentions)
Anti cd11c, by BioLegend (40 mentions)
Anti f4 80, by BioLegend (32 mentions)
Anti cd44, by BioLegend (26 mentions)
Lsrfortessa, by BD (20 mentions)
Anti ly6g, by BioLegend (20 mentions)
Anti nk1, by BioLegend (19 mentions)
Anti cd62l, by BioLegend (17 mentions)
Anti cd86, by BioLegend (17 mentions)
Anti cd19, by BioLegend (17 mentions)
Anti cd90, by BioLegend (17 mentions)
Anti cd31, by BioLegend (16 mentions)
Propidium iodide, by Thermo Fisher Scientific (15 mentions)
Anti cd69, by BioLegend (14 mentions)
Anti ly6c, by BioLegend (14 mentions)
Anti ly6g, by BD (12 mentions)
Anti b220, by BioLegend (12 mentions)
228 protocols using anti cd45
1
Isolation of Murine Lung Cell Populations
2
Isolation of Murine Lung Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BALB/c mice were perfused with 5 ml of HBSS through the pulmonary artery via the right ventricle and instilled with 4.5 U/ml of elastase (Roche Diagnostics) via tracheal cannula. The lung lobes were then minced in 100 U/ml DNase I (Aladdin). Cells in suspension were subsequently filtered through a 40 μm nylon mesh and then sorted by FACS. Anti-CD45 (BioLegend, 157607, 1:200 dilution) and anti-CD3 (BioLegend, 100209, 1:200 dilution) antibodies were used for the isolation of T cells. Anti-CD45 and anti-CD19 (BioLegend, 152407, 1:200 dilution) antibodi were used for isolation of B cells. Anti-CD45 and anti-NK-1.1 (BioLegend, 108703, 1:200 dilution) antibody were used for isolation of NK cells. Anti-CD45 and anti-F4/80 (BioLegend, 123105, 1:200 dilution) antibodies were used for the isolation of macrophages. Anti-CD45 and anti-Ly6G (BioLegend, 127627, 1:200 dilution) antibodies were used for the isolation of neutrophils. Anti-EpCAM (BioLegend, 324215, 1:200 dilution) and anti-SP-C (abclonal, A1835, 1:500 dilution) antibodies were used for the isolation of alveolar epithelial cells. anti-CD31 (BioLegend, 102409, 1:200 dilution) antibody was used for the isolation of endothelial cells. Anti-S100A4 (abcam, ab197896, 1:500 dilution) antibody was used for the isolation of fibroblasts.
3
Multicolor Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedures used in this experiment have been published previously [25 (link),26 (link)]. Immune cells were evaluated in each experiment and treated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany), stained with fluorescence-labeled antibodies as indicated, and incubated for 20 min at 4 °C in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin. The following mouse antibodies were purchased from BioLegend (San Diego, CA, USA): anti-CD3 (labeled with FITC, PE, and APC-Cy7), anti-CD4 (PE), anti-CD8 (FITC and APC-Cy7), anti-CD11b (APC-Cy7), anti-CD11c (BV421), anti-CD40 (APC), anti-CD44 (PE), anti-CD45 (PerCP-Cy5.5), anti-CD45.1 (Pacific Blue), anti-CD45.2 (Pacific Blue), anti-CD62L (PerCP), anti-CD69 (PE), anti-CD80 (PerCP-Cy5.5), anti-CD86 (PE), anti-CD103 (PE-Cy7), anti-CD127 (BV510), and anti-NK1.1 (PerCP-Cy5.5.). We also used 7-AAD (BioLegend) to stain dead cells for the evaluation of cell viability. The stained cells were subjected to FACS Verse for data acquisition (Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
4
Comprehensive Immunophenotyping of T Cells
5
Isolation and Flow Cytometry of Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry analysis was performed on a LSRII or LSRFortessa (BD Biosciences). To isolate neutrophils from the feet, tissue was diced and incubated at 37°C for 45 min with shaking at 200 rpm (TissueLyserII, Qiagen), in DMEM containing 1 mg/mL Collagenase Type IV (Worthington Biochemical). Cells were then strained through a 70 μM strainer and washed twice in phosphate buffered saline (PBS). Neutrophils from bone marrow and feet were sorted on a FACSAria III (BD Biosciences). FcγR were blocked using TruStain FcX (BioLegend). Data were analyzed using FlowJo software (BD Biosciences). The following reagents were used: Zombie-NIR fixable viability kit (BioLegend), anti-B220 (eBioscience;RA36B2), anti-CD3 (WEHI;KT3.1.1), anti-CD4 (WEHI;GK1.5), anti-CD8 (WEHI;53.6.7), anti-CD11b (BioLegend;M1/70), anti-CD16/32 (WEHI;24G2), anti-CD34 (eBioscience;RAM34), anti-CD45.1 (BioLegend;A20), anti-CD45.2 (BioLegend;104), anti-CD48 (BD Bioscience;C2), anti-CD127 (eBioscience;A7R34), anti-CD135 (BioLegend;A2F10), anti-CD150 (BioLegend;TC15–12F12.2), anti-cKit (WEHI;ACK4), anti-Gr1 (WEHI;RB6–8C5), anti-Ly6G (BD;1A8), anti-Sca-1 (WEHI;Ly6A/E), anti-Siglec-F (Fisher;1RMN44N), AnnexinV-AF647 (BioLegend), and propidium iodide (Sigma).
6
Isolation and Flow Cytometry of Neutrophils
7
Detailed Antibody Characterization for Immunoblotting and Flow Cytometry
8
Multicolor Flow Cytometry for Immune Cell Analysis
9
Multicolor FACS and IHC Antibody Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A full list of antibodies is provided in the Key Resource table (Table 1 ). The following antibodies were used for FACS analysis: Anti-mouse/human CD45R/B220, anti-CD23, anti-CD21/CD35 (CR2/CR1), anti-CD93 [AA4.1], anti-mouse IgM, anti-IgD, anti-CD45.1, anti-CD45.2, anti-CD3ε, anti-Ly-6G/Ly-6C(Gr-1), anti-CD11b, anti-TCRb, and anti-CD5 were all purchased from Biolegend. Anti-Mouse CD19 was purchased from BD Biosciences. Rabbit Anti-Ki67 (Novocastra), rabbit anti-cleaved caspase 3 (Cell signaling), rabbit anti-Phospho-Histone H3 (Ser10) (Cell signaling), rat anti-Pax5 (Biolegend), Guinea pig anti-Cytokeratin 8+18 antibody, and FITC rat Anti-mouse/human CD45R/B220 (BioLegend) were used for fluorescence immunohistochemistry. Anti-rabbit Alexa Fluor Cy3 (Jackson ImmunoResearch), anti-rabbit Alexa Fluor 647 (Jackson Immunoresearch), anti-rat Cy3 (Jackson Immunoresearch), and Alexa Fluor anti-guinea pig 647 secondary antibodies were used for in fluorescence immunohistochemistry. Notch2 (D76A6) XP (Cell signaling) and HRP-linked rabbit IgG (GE healthcare) secondary antibodies were used for western blot analysis.
10
Multiparametric Flow Cytometry and Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following fluorochrome-conjugated antibodies and reagents were used: anti-CD3 (145–2C11, 5 μg/ml for flow cytometry and 10μg/ml for microscopy), anti-CD4 (RMA 4–5, 1 μg/ml), anti-CD8 (53–6.7, 2.5 μg/ml), anti-CD44 (IM7, 2.5μg/ml), anti-B220/CD45RO (RA3–6B2, 2.5 μg/ml), anti-CD69 (H1.2F3, 2μg/ml), anti-NK1.1 (PK136, 0.5 mg/ml), anti-I-A/I-E (M5/114.15.2, 0.2 mg/ml), anti-CD11c (N418, 0.2 mg/ml), anti-CD14 (Sa14–2, 0.2 mg/ml), anti-CD45.1 (A20, 0.5 mg/ml), anti-CD45.2 (104, 0.5 mg/ml), anti-IL-17A (TC11–18H10.1, 0.2 mg/ml), anti-IFN-γ (XMG1.2, 0.2 mg/ml), streptavidin (2.5 μg/ml for flow cytometry and 1.25μg/ml for microscopy) (all from BioLegend). Fluorochrome-conjugated anti-Lyve1 (ALY7, 2.5 μg/ml), anti-FoxP3 (FJK-16 s, 0.5 mg/ml) were from eBioscience. Anti-S1PR1 (clone 713412) was from R&D Systems. CD4 (RMA 4–5, 0.2 mg/ml) was from BD Biosciences. Propidium iodide (0.75μg/ml), DAPI (0.125μg/ml) or LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher L23105) was used to exclude dead cells.
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!