Seven and 14 days after implant, epididymal fat pads were harvested following euthanasia and washed in ice cold PBS (Sigma). Fat pads were minced, digested in collagenase (Liberase TL, Roche), and passed through a 100 μm filter. The stromal vascular fraction was harvested by centrifugation, washed in MACS buffer (PBS, 0.5 mM EDTA, 30% BSA), and incubated with anti-CD16/32 prior to adding an antibody cocktail against extracellular antigens. The following antibodies were purchased from Biolegend: anti-CD45 clone 30-F11, anti-Ly6G clone 1A8, anti-F4/80 clone BM8, anti-NK1.1 clone PK136, anti-CD19 clone 6D5, anti-CD11b clone M1/70, anti-CD3 clone 17A2, and Trustain fcX (anti-CD16/32) clone 93. The following isotype controls were also purchased from Biolegend: mouse IgG2a clone MOPC-173; rat IgG2a clone RTK2758; rat IgG2b clone RTK4530. After antibody incubation, cells were washed, fixed, and analyzed using a FACS Aria flow cytometer (BD Biosciences). The number of CD45 cells in each flow cytometry sample was calculated using Bang’s labs Flow Cytometry Absolute Count Standard, which was added prior to data acquisition. FlowJo software (Treestar) was utilized to compensate and analyze data. FMOs with isotype controls were used to determine specific antibody signal. The gating scheme used in the flow cytometry analysis is depicted in Figure S3 .
Anti cd45 clone 30 f11
Manufactured by BioLegend
Sourced in United States
Anti-CD45 (clone 30-F11) is a monoclonal antibody that binds to the CD45 antigen. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b clone m1 70, by BioLegend (7 mentions)
Anti f4 80 clone bm8, by BioLegend (5 mentions)
Anti ly6g clone 1a8, by BioLegend (4 mentions)
Anti cd11c clone n418, by BioLegend (4 mentions)
Percoll, by GE Healthcare (4 mentions)
Anti ly6c clone hk1, by BioLegend (3 mentions)
Anti siglecf clone e50 2440, by BD (3 mentions)
Anti epcam clone g8.8, by BioLegend (3 mentions)
Catalog no l10119, by Thermo Fisher Scientific (3 mentions)
Liberase tl, by Roche (2 mentions)
Anti nk1.1 clone pk136, by BioLegend (2 mentions)
Anti cd3 clone 17a2, by BioLegend (2 mentions)
Anti cd16 32, by BioLegend (2 mentions)
Anti b220 clone ra3 6b2, by BioLegend (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Cd16 cd32 fc block clone 93, by BioLegend (2 mentions)
Anti b220 cd45r clone ra3 6b2, by Thermo Fisher Scientific (2 mentions)
Anti ly 6g clone 1a8, by Thermo Fisher Scientific (2 mentions)
Anti cd3ε clone 145 2c11, by BioLegend (2 mentions)
Syrian hamster igg, by Jackson ImmunoResearch (2 mentions)
27 protocols using anti cd45 clone 30 f11
1
Epididymal Fat Pad Characterization
2
Tumor Cell Isolation and Gene Expression
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Cell suspension (described above) was stained with Zombie Fixable Viability Kit followed by incubation with anti-CD16/32 and staining with anti-CD45 (clone 30-F11) and anti-CD31 (clone 390); all from Biolegend. At least 50,000 tumor cells (CD45neg,CD31neg,GFP+) were sorted per sample. RNA was extracted using RNeasy Plus Mini Kit (Qiagen) and cDNA was synthesized using Omniscript RT Kit (Qiagen). qPCR was performed with KAPA SYBR FAST quantitative PCR (qPCR) Master Mix (KAPA Biosystems) and analyzed in a CFX96 Touch Real-Time PCR Detection System (Biorad).
3
Isolation and Characterization of Immune Cells
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After collection of neat supernatant for MV harvesting, the remaining adherent cells were dissociated from each well using Versene solution (Thermo Fisher Scientific, Waltham, MA, USA) for 7 min at 37°C. Cell pellets were isolated by centrifugation and incubated with a fluorescence-conjugated antibody cocktail containing anti-CD45 (clone 30-F11; BioLegend), anti-CD11b (M1/70; BD Biosciences, San Jose, CA, USA), anti-TNF (MP6-XT22; BioLegend), and its corresponding isotype control, IgG1κ (MOPC-21; BioLegend), for 30 min at 4°C.
4
Comprehensive Immune Profiling of Tumor Microenvironment
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Tumors enzymatically digested by ± 2.3 Wunsch units / ml Liberase TL (Roche, Basel, Switzerland) and tumor draining lymph nodes were passed through 70 μm cell strainers. The generated single cell suspensions were stained with the live/dead marker Zombie Aqua (Biolegend, San Diego, CA, USA) and subsequently blocked for unspecific binding to CD16/32 (TruStain fcX, Biolegend). In order to investigate different myeloid cell populations and endothelial cell activation the following antibodies were diluted in FACS buffer (1% FCS, 0.02% NaN3 and 3 mM EDTA in PBS): anti-CD11b (clone M1/70, Biolegend), anti-CD11c (clone N418, Biolegend), anti-B220 (clone RA3-6B2, Biolegend), anti-CD86 (clone GL-1, Biolegend), anti-CD45 (clone 30-F11, Biolegend), anti-Gr1 (clone RB6-8CS, Biolegend), anti-Ly6C (clone HK1.4, Biolegend), Ly6G (clone 1A8, Biolegend), ICAM-1 (clone YN1/1.7.4, Biolegend), VCAM-1 (clone MVCAM.A, Biolegend) and CD31 (clone MEC 13.3, BD Biosciences, Franklin Lakes, NJ, USA). Samples were washed with FACS-buffer and analyzed in a FACSCanto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed with FlowJo software (TreeStar, Ashland, OR, USA).
5
Comprehensive Lung Cell Characterization
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Lung tissue was harvested as indicated and single cell suspension were prepared using a metal mesh. Absolute cell numbers were counted with a Neubauer chamber. Single-cell suspensions of the lungs respectively collected BAL fluid cells were incubated with CD16/CD32 Fc block (clone 93, BioLegend, 101310) to inhibit unspecific antibody binding. For flow cytometry, cells were stained with the following antibodies: anti-B220/CD45R (clone RA3-6B2, eBioscience, 45-0452), anti-CD3ε (clone 145-2C11, BioLegend, 100320), anti-CD11b (clone M1/70, BioLegend, 101206), anti-Ly-6G (clone 1A8, eBioscience, 17-9668), anti-CD45 (clone 30-F11, BioLegend, 103137), anti-CD11c (clone N418, Biolegend, 117333) and anti-SiglecF (clone E50-2440, BD, 562681). To exclude dead cells from the analysis, the samples were labeled with the Fixable Viability Dye eFluor 780 (eBioscience, 65-0865).
6
Enumerating Intracellular Mycobacteria in Macrophages
7
Isolation of Murine Corneal Cells
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Adult mouse corneas anterior to the limbus were excised and incubated in 20 mM EDTA-PBS for 20 min, followed by chopping with scissors into tiny pieces and incubated with 0.1% type IV Collagenase for 1 h to yield single-cell suspensions. The samples were then incubated with anti-CD16/32 (2.4G2, Catalog no. 553141, BD Pharmingen™, San Diego, CA), for 5 min at room temperature and subsequently stained with surface markers anti-CD45 (clone 30-F11, Catalog no. 103138, BioLegend), anti-CD11b antibody (Clone M1/70, Cat. No. 552850, BD Pharmingen™, SanDiego, CA), anti-MRC1 (Clone Y17-505, Catalog no. 568808, BD Pharmingen™, SanDiego, CA), and anti-Mer (Clone 108,928, Catalog no. FAB5912G, R&D bio-techne, Eugene, OR) antibodies. Additionally, live/dead discrimination staining was performed using LIVE/DEAD™ Fixable Near-IR dye (Catalog. no. L10119A, Thermo Fisher Scientific Inc.). The gating strategy was as follows: lymphocytes were identified by forward-scatter area (FSC-A) and side scatter area (SSC-A) gates, followed by two singlets gates (FSC-A vs. FSC-W and SSC-A vs. SSC-W) followed by live/dead identification using the infra-red fluorescent viability dye.
8
Tumor-Infiltrating Immune Cell Profiling
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Single-cell suspensions of transplanted tumors were obtained by digestion with 1 mg/ml Collagenase I (Sigma) and 1 mg/ml Dispase II (Roche) in RPMI containing 5 % FBS for 45 min at 37°C under shaking condition. The cell suspension was passed through a 70 μm nylon mesh and centrifuged. After red blood cell lysis, Fc receptors were blocked with anti-mouse FcR antibody (clone 93, Biolegend) for 15 min at a 1:100 dilution on ice. Then, cells were stained with anti-CD45 (clone 30-F11, Biolegend), CD11b (clone M1/70, Biolegend), Gr-1 (clone RB6-8C5, Biolegend), F4/80 (clone BM8, Biolegend), PDGFRα (clone APA5; Biolegend), CD206 (clone C068C2, Biolegend), CD11c (clone N418, Biolegend) and MHC II (I-A/I-E, clone M5/114.15.2, Biolegend) antibodies for 30 min at a 1:100 dilution on ice. For analysis of Treg cells in the tumor, cells were stained with anti-CD4 (clone GK1.5, Biolegend, 1:100), Foxp3 (clone FJK-16s, eBioscience, 1:50) antibodies using a Foxp3 staining buffer kit following the instructions (eBioscience). Samples were acquired on a Canto II flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software.
9
Flow Cytometry Analysis of Intestinal Epithelial Cells and Rotavirus Infection
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Dissociated cells were collected and stained for flow cytometry. Cells were stained with Zombie Aqua viability dye (BioLegend), Fc receptor-blocking antibody (CD16/CD32; BioLegend), anti-EpCAM (clone G8.8; BioLegend), and anti-CD45 (clone 30-F11; BioLegend). For analysis of murine rotavirus infection, cells were stained with anti-rotavirus (polyclonal; ThermoFisher, #PA1-7241) followed by goat anti-rabbit secondary (ThermoFisher). All data were analyzed using FlowJo software (BD Biosciences). Gates were set based on unstained and single-fluorophore stains. IECs were selected by gating on live, EpCAM-positive, CD45-negative cells. Gates for murine rotavirus infection were set based on naïve samples.
Where indicated, dissociated cells were enriched using MojoSort Mouse anti-APC Nanobeads (BioLegend, #480072) after flow cytometry staining for anti-EpCAM and anti-CD45 with APC fluorophores by following manufacturer protocols.
Where indicated, dissociated cells were enriched using MojoSort Mouse anti-APC Nanobeads (BioLegend, #480072) after flow cytometry staining for anti-EpCAM and anti-CD45 with APC fluorophores by following manufacturer protocols.
10
Comprehensive Lung Cell Characterization
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