Single cell suspensions of TECs were obtained collagenase (Collagenase D type IV, Worthington) and dispase (Roche) enzymatic dissociation as previously described [78 (link)] and thymocytes were obtained by pressing the thymus through a 70μm strainer (Fisher). Cells were stained with fluorochrome-conjugated antibodies in FACS buffer (PBS pH 7.2, 0.005M EDTA, 2% FBS) for 20 minutes on ice and washed. Propidium iodide (Invitrogen) was added (0.5 μg/ml) to each sample prior to analysis to exclude dead cells. Anti-CD326 (clone G8.8), anti-I-A/I-E (clone M5/114.15.2), anti-CD25 (clone PC61) and anti-CD117 (clone 2 B8) were purchased from Biolegend. Anti-CD44 (clone IM7), anti-CD8α (clone 53–6.7), anti-CD45 (clone 30-F11) and anti-Bcl-2 (clone 3F11) were purchased from eBioscience. Biotinylated anti-Ly51, anti-pStat3 (clone 4/p-Stat3 pY705) and anti-total Stat3 (clone M59-50) were purchased from BD Biosciences. Anti-CD4 (clone RM4-5) and Streptavidin Qdot 655 was purchased from Invitrogen. To exclude erythrocytes, granulocytes, dendritic cells, macrophages and NK cells from the thymocyte analyses, the following antibodies were purchased from eBioscience: TER-119, CD11c (cloneN418), CD11b (clone M1/70), NK-1.1 (clone PK136) and Ly-6G (clone RB6-8C5). Flow cytometry was performed on a Becton Dickenson FACS Aria II and data were analyzed using FlowJo software (Tree Star).
Anti cd45 clone 30 f11
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-CD45 (clone 30-F11) is a mouse monoclonal antibody that recognizes the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells. This antibody clone can be used for the identification and enumeration of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b clone m1 70, by Thermo Fisher Scientific (7 mentions)
Lsrfortessa, by BD (5 mentions)
Anti cd80 clone 16 10a1, by BioLegend (5 mentions)
Streptavidin pe cy7, by Thermo Fisher Scientific (3 mentions)
Collagenase dispase, by Roche (3 mentions)
Dnase 1, by Roche (3 mentions)
Anti epcam1 clone g8, by Thermo Fisher Scientific (3 mentions)
Anti ly51 clone 6c3, by BioLegend (3 mentions)
Cd104 clone 346 11a, by BioLegend (3 mentions)
Facsaria fusion 1 cell sorter, by BD (3 mentions)
Anti mhc 2 clone m5 114, by Thermo Fisher Scientific (3 mentions)
Biotinylated uea 1, by Vector Laboratories (3 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (3 mentions)
Anti cd3 clone 145 2c11, by BioLegend (3 mentions)
Anti cd25 clone pc61, by BioLegend (3 mentions)
Anti ly6c clone hk1, by BioLegend (3 mentions)
Fixable viability dye, by Thermo Fisher Scientific (3 mentions)
Facscanto 2, by BD (2 mentions)
Anti gr 1 clone rb6 8c5, by BioLegend (2 mentions)
Anti cd4 clone rm4 5, by Thermo Fisher Scientific (2 mentions)
23 protocols using anti cd45 clone 30 f11
1
Isolation and Analysis of Thymic Epithelial Cells
2
Multi-color Flow Cytometry Gating
3
Isolation and Analysis of Lung Immune Cells
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After the removal of the left lobe of mice lung, the tissue was chopped and digested with 15 mg/mL collagenase (Type D; Roche Diagnostics) and 25 μg/mL DNase (Type 1; Roche Diagnostics) in 4 mL RPMI + 10% CFS for 1 h at 37 °C with agitation. Lung was then passed through a 70 μm sieve (BD Bioscience). The resulting cell suspension was washed, red blood cells were lysed, and the remaining cells were resuspended in 1 mL RPMI. To analyze cell populations, the cell suspension was labeled using monoclonal antibodies anti-CD45 (clone 30-F11) (1:100) anti-Ly6G (clone 1A8) (1:400), anti-F4/80 (clone BM8) (1:100), anti-CCR5 (HM-CCR5 (7A4)) (1:200) from eBioscience for 30 min. All data was acquired by flow cytometry (FACSCalibur; BD Biosciences PharMingen) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). Gating strategies for the identification of neutrophils and macrophages in these experiments are shown in Figure S2 .
4
Quantification of Alveolar Macrophages and Neutrophils
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BALF and lung cells were stained at 4°C in RPMI 1640 medium containing 1% FBS after FcγRII/III blocking with anti-mouse CD16/CD32 (clone 93; eBioscience). Surface staining was performed with antibodies purchased from eBioscience (anti-CD45; clone 30-F11; anti-Ly6G (Gr-1), clone RB6-8C5; anti-CD11c, clone N418; and anti-CD11b, clone M1/70). Alveolar macrophages (CD11b−CD11c+) and neutrophils (CD11b+Gr-1+) were analysed.
For intracellular staining, lung and spleen cells were stimulated with heat-killed bacteria at MOI (multiplicity of infection) 100 for 16 h at 37°C with Golgi block added on the last 5 hours. Cells were then surface-stained with anti-CD4 (clone RM4-5; eBioscience) and anti-γδTCR (clone GL-3; eBioscience) and followed by permeabilization with Cytofix-Cytoperm solution (BD Pharmingen). Then, the cells were stained with anti-IL-17A (clone 17B7; eBioscience) and anti-IFN-γ (clone XMG1.2; eBioscience). All samples were analysed with FACSCalibur. Data were analysed with FlowJo software.
For intracellular staining, lung and spleen cells were stimulated with heat-killed bacteria at MOI (multiplicity of infection) 100 for 16 h at 37°C with Golgi block added on the last 5 hours. Cells were then surface-stained with anti-CD4 (clone RM4-5; eBioscience) and anti-γδTCR (clone GL-3; eBioscience) and followed by permeabilization with Cytofix-Cytoperm solution (BD Pharmingen). Then, the cells were stained with anti-IL-17A (clone 17B7; eBioscience) and anti-IFN-γ (clone XMG1.2; eBioscience). All samples were analysed with FACSCalibur. Data were analysed with FlowJo software.
5
Cardiac Immune Cell Infiltration Analysis Post-Myocardial Infarction
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For the evaluation of infiltration of immune cells into hearts post-MI, the excised hearts were minced with fine scissors into 1–2 mm3 pieces. The minced heart tissue was transferred into a tube containing enzyme solution (200 units/mL collagenase II (Worthington, Lakewood, NJ, USA), 500 units/mL hyaluronidase type IV-S (Sigma-Aldrich, St. Louis, MO, USA), and 100 units/mL DNase I (Takara Biotec, Kusatsu, Japan) and dissociated using a gentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The dissociated cells were stained directly using fluorochrome-conjugated mouse-specific antibodies and analyzed with a FACSVerse™ instrument (BD Biosciences, San Jose, CA, USA) using FlowJo version 10.2 software (BD Biosciences). Cells that did not stain with 7-AAD (TONBO Biosciences, San Diego, CA, USA) were deemed viable. The antibodies used were as follows: anti-CD45 (clone 30-F11; eBioscience, San Diego, CA, USA), anti-Ly6G (clone RB6-8C5; TONBO Biosciences), anti-CD11b (clone M1/70; eBioscience), anti-F4/80 (clone BM8.1; TONBO Biosciences), and anti-CD206 (clone C068C2; BioLegend). To evaluate apoptosis in vitro, HL-1 cells were stained with 7-AAD and FITC Annexin V (BD Biosciences).
6
Thymic Epithelial Cell Isolation and Analysis
7
Immunophenotyping of Mouse Stem Cells
8
Cardiac Cell Isolation and Analysis
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The excised hearts were minced with fine scissors into 1‐2 mm3 pieces. The minced heart tissue was transferred into a tube containing enzyme solution (200 units/mL collagenase II [Worthington], 500 units/mL hyaluronidase type IV‐S [Sigma] and 100 units/mL DNase I [Takara Biotec]) and dissociated using the gentleMACS™ Dissociator (Miltenyi Biotec). For flow‐cytometric analysis, the dissociated cells were stained directly by using fluorochrome‐conjugated mouse‐specific antibodies and analysed with a FACSVerse™ instrument (BD Biosciences) using FlowJo version 10.2 software (Tree Star). Cells that did not stain with 7‐AAD (Tonbo Biosciences) were deemed viable. The antibodies used were as follows: anti‐CD45 (clone 30‐F11; eBioscience), anti‐c‐Kit (clone 2B8; BioLegend), anti‐Sca‐1 (clone E13‐161.7; BioLegend), anti‐Ly6G (clone RB6‐8C5, TONBO Biosciences), anti‐CD11b (clone M1/70, eBioscience), anti‐F4/80 (clone BM8.1; Tonbo Biosciences), anti‐CD206 (clone C068C2, BioLegend) and anti‐MAIR‐II (clone TX52; BioLegend).
9
Multiparametric Immune Profiling of BALF
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BALF cells were stained at 4°C in PBS containing 1% FBS after FcγRII/III blockade with anti-mouse CD16/CD32 (clone 93; eBioscience). Surface staining was performed with antibodies purchased from eBioscience (anti-CD45, clone 30-F11; anti-CD11b, clone M1/70; anti-CD11c, clone N418; anti-Ly6G (Gr-1), clone RB6-8C5; anti-CD3, clone 145-2C11; anti-CD4, clone RM4-5; and anti-CD44, clone IM7). For Treg staining, cells were surface-stained with anti-CD4 and anti-CD25 (clone PC61.5) and followed by permeabilization with the Foxp3/Transcription Factor Buffer Staining Set (eBiosciences). Then the cells were stained with anti-foxp3 (clone FJK16s) antibody. For intracellular staining, cells were surface-stained with anti-CD4 and anti-CD44, followed by permeabilization with the Fixation/Permeabilization Solution Kit (BD). Then the cells were stained with anti-IFN-γ (Clone XMG1.2), anti-IL-17 (Clone eBio17B7), and anti-IL-4 (Clone 11B11). The apoptotic status of lung cells was detected with the Apoptosis Detection kit (BD Pharmingen) following the instructions of the manufacturer. In this method, apoptosis was examined by the Annexin V/7-AAD double staining. All samples were analysed with FACSCalibur and FACSFortessa. Data were analysed with FlowJo.
10
Isolation and Flow Cytometry Analysis of Thymic Epithelial Cells
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For TEC analysis, single-cell suspensions were generated by digesting thymic lobes with collagenase Dispase (2.5 mg/ml; Roche) and DNase 1 (40 mg/ml; Roche). CD45− cells were enriched by the depletion of CD45+ cells using anti-CD45 beads and LS columns (Miltenyi Biotec). The following Abs were used for TEC analysis: anti-CD45 clone 30-F11 (eBioscience), anti-EpCAM1 clone G8.8 (eBioscience), anti-Ly51 clone 6C3 (BioLegend), anti–MHC II clone M5/114.15.2 (eBioscience), anti-CD80 clone 16-10A1 (BioLegend), CD104 clone 346-11A (BioLegend), and anti–MHC I 28-14-8. Biotinylated UEA-1 (Vector laboratories) was detected using streptavidin PECy7 (eBioscience). Cells were analyzed using a LSR Fortessa (Becton Dickinson) with data analysis carried out using FlowJo v10 (Becton Dickinson). For cell sorting, TEC subsets were identified using the earlier Abs and isolated using a FACSAria Fusion 1 cell sorter (Becton Dickinson). The sorting strategy for the different TEC subsets was as follows: Cxcl12DsRed+ cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed+; CXCL12DsRed− cTEC, CD45−EpCAM1+UEA1−Ly51+CXCL12DsRed−; mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−; mTEChi, CD45−EpCAM1+UEA+Ly51−CD80+MHC II+; CD104+ mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104+; and CD104− mTEClo, CD45−EpCAM1+UEA1+Ly51−CD80−MHC II−CD104−.
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