For the extraction of CNS infiltrating cells, spinal cords were harvested after intensive transcardial perfusion with DPBS. CNS tissues were mechanically homogenized in DPBS, layered on a 30–50% Percoll (Merck KGaA) gradient and centrifuged for 30 min at 1200× g without using the brake. Calibrite beads (BD) were added before washing and staining for quantification of cell numbers isolated from the interphase. To assess the number of infiltrated platelets, CNS homogenates were centrifuged at 250× g for 5 min at RT to receive platelet-rich supernatant. This supernatant was stained for CD41 (clone MWReg30, BioLegend, San Diego, CA, USA), as well as CD61 (clone 2C9.G2, BioLegend), and fixed with 1% PFA before evaluation. CNS infiltrating cells were stained with fluorochrome-conjugated antibodies from BioLegend for CD3 (clone 17A2), CD4 (clone GK1.5), CD8a (clone 53-6.7), CD11b (clone M1/70), CD45R/B220 (clone RA3-6B2) and CD45 (clone 30-F11). Corresponding isotype controls were used for all stainings and for blocking the Fc receptor binding; cells were preincubated with an anti-CD16/CD32 antibody (BioLegend) for 5 min on ice. The stained samples were analyzed using a multi-color flow cytometer (Gallios, Beckman Coulter, Krefeld, Germany) and Kaluza software (Beckman Coulter). Cell doublets were excluded to ensure single cell counting.
Cd45 clone 30 f11
Manufactured by BioLegend
Sourced in United States
CD45 (clone 30-F11) is a mouse monoclonal antibody that recognizes the pan-leukocyte marker CD45. CD45 is a transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated hematopoietic cells and is essential for the development and activation of T and B cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b clone m1 70, by BioLegend (17 mentions)
Ly6g clone 1a8, by BioLegend (7 mentions)
Cd11c clone n418, by BioLegend (7 mentions)
Ly6c clone hk1, by BioLegend (7 mentions)
Cd11b clone m1 70, by BD (7 mentions)
Cd3 clone 17a2, by BioLegend (6 mentions)
F4 80 clone bm8, by BioLegend (6 mentions)
Cd4 clone gk1, by BioLegend (5 mentions)
Cd8 clone 53 6, by BioLegend (5 mentions)
Cd64 clone x54 5 7, by BioLegend (5 mentions)
Facsaria, by BD (4 mentions)
Cd19 clone 6d5, by BioLegend (4 mentions)
Cd3e clone 145 2c11, by BioLegend (4 mentions)
Ionomycin, by Merck Group (4 mentions)
Mhcii clone m5 114, by BioLegend (4 mentions)
Foxp3 clone fjk 16s, by Thermo Fisher Scientific (4 mentions)
Cd206, by BioLegend (4 mentions)
Cd86 clone gl 1, by BioLegend (4 mentions)
Tcrβ clone h57 597, by BioLegend (3 mentions)
Pd l1 clone 10f 9g2, by BioLegend (3 mentions)
62 protocols using cd45 clone 30 f11
1
Extraction and Quantification of CNS-Infiltrating Cells
2
FACS Purification of Lung-Resident Immune Cells
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For FACS purification of lung-resident populations, we used antibodies against CD3ɛ (clone 145-2C11), CD4 (clone RM4-5), CD11b (clone M1/70), Ly6G (clone 1A8), CD19 (clone 6D5), CD45 (clone 30-F11), TCRβ (clone H57-597), and TCRγ/δ (clone GL3) from BioLegend. 7AAD was from BD Pharmingen. Single-cell suspensions were generated from lungs of two or three C57Bl/6J mice with a lung-dissociation kit (Miltenyi Biotec). Single-cell suspensions were incubated with CD90.2 MicroBeads (Miltenyi Biotec) and separated into CD90.2-positive and CD90.2-negative fractions. Both fractions were stained on ice with surface antibodies and live/dead marker 7AAD, and sorted on a BD FACSAria (BD Biosciences). Different cell types were identified through the following gating strategies: B cells (7AAD−CD45+CD19+) and neutrophils (7AAD−CD45+CD11b+Ly6G+) were sorted from the preenriched CD90.2− cell fraction, whereas CD4+ T cells (7AAD−CD45+CD3+TCRβ+CD4+) and TCRγδ T cells (7AAD−CD45+CD3+TCRβ−TCRγδ+) were sorted from the preenriched CD90.2+ cell fraction.
3
Analyzing Immune Cell Populations in Murine Tissues
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We performed FACS analysis of BM chimera using CD45.1 and CD45.2
antibodies (Biolegend). Acquisition was performed with a BDBioscienes
FACSCalibur and analysis with FlowJo software. For the FACS analysis of BAT of
the THcre:dTomatof/f:CX3CR1gfpmice, tissue was collected, dissected by scissors and then incubated for 30 min
with DMEM medium (Beit Ha’emek, Israel) containing 1 mg/ml collagenase-2
(Sigma-Aldrich), 2% BSA (Sigma-Aldrich) and 12.5 mM HEPES buffer
(Beit-Ha’emek, Israel). The resulting cell suspension was filtered
through a 70 µm mesh and erythrocytes were removed by ACK lysis.
Following cell suspension, cells were incubated in FACS buffer (PBS with 1% BSA,
2 mM EDTA and 0.05% sodium azide) in the presence of staining antibody.
Antibodies used for staining were CD45 (clone 30F11, Biolegend), CD14 (clone
Sa2-8, Biolegend) and F4/80 (clone BM8, Serotech).
antibodies (Biolegend). Acquisition was performed with a BDBioscienes
FACSCalibur and analysis with FlowJo software. For the FACS analysis of BAT of
the THcre:dTomatof/f:CX3CR1gfpmice, tissue was collected, dissected by scissors and then incubated for 30 min
with DMEM medium (Beit Ha’emek, Israel) containing 1 mg/ml collagenase-2
(Sigma-Aldrich), 2% BSA (Sigma-Aldrich) and 12.5 mM HEPES buffer
(Beit-Ha’emek, Israel). The resulting cell suspension was filtered
through a 70 µm mesh and erythrocytes were removed by ACK lysis.
Following cell suspension, cells were incubated in FACS buffer (PBS with 1% BSA,
2 mM EDTA and 0.05% sodium azide) in the presence of staining antibody.
Antibodies used for staining were CD45 (clone 30F11, Biolegend), CD14 (clone
Sa2-8, Biolegend) and F4/80 (clone BM8, Serotech).
4
Immunofluorescent Staining of Tumor Cryosections
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Tumor samples were fixed in 10% buffered formalin solution and embedded in optimal cutting temperature (OCT) (Tissue-Tek) medium. Cryosections (8–10 mm) were stained for immunofluorescence. Immunofluorescent stains were applied using BS-I Isolectin B4 Biotin Conjugate (Sigma-Aldrich), monoclonal mouse CD8 (clone 53-6.7, eBioscience) and CD45 (clone 30-F11, Biolegend). Briefly, tumor sections were dried at room temperature (RT), washed three times with cold PBS and blocked with 10% BSA for 30 minutes. Samples were incubated for 2 hours at RT with primary antibodies, washed and incubated for 1 hour at RT with the secondary antibody. Antibodies were used between 5–10 μg/ml. After washing with cold PBS three times, tissues were covered with Prolong Gold anti-fade reagent with DAPI (Invitrogen). Images of antibody-stained sections were acquired using the Sp5 Tandem Scanner confocal microscope running LAS AF software (Leica Microsystems, Barcelona, Spain).
5
Comprehensive Tumor Cell Phenotyping
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Totally, 1.5 million cells from tumors in mTmG mice were stained following the BD Cytofix/Cytoperm Fixation/Permeabilization microplate staining protocol (BD Biosciences), using the following antibodies: CD16/32 Fc Block (clone 2.4G2, BD Biosciences cat. 553142, 1:100), CD45 (clone 30-F11, BioLegend cat. 103127, 1:350), PD-L1 (clone 10F.9G2, BioLegend cat. 124313, 1:100), CD11b (clone M1/70, BioLegend cat. 101223, 1:100), CD11c (clone N418, BioLegend cat. 117323, 1:100). Cell lines were stained with PD-L1 (clone 10F.9G2, BioLegend cat. 124307, 1:100). Dead cells were excluded using Ghost Dye Violet 510 Dead Cell Stain Kit (Tonbo Biosciences) according to manufacturer protocol, and compensation was calibrated with single-color controls on Ultracomp eBeads (Invitrogen). Cells were quantified using a Gallios 561 cytometer (Beckman Coulter) and analyzed using Kaluza analysis software (Beckman Coulter) with fluorescence-minus-one controls to verify gating.
6
Multiparametric Immune Phenotyping of Cells
7
Multiparameter Flow Cytometry of Immune Cells
8
Detecting ERV Envelope on Lymphocytes
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ERV envelope on lymphocytes was detected as previously described (42 (link); b)using an anti-MLV envelope antibody clone 83A25 (kindly provided by Leonard Evans, NIH) (41 (link)). The following antibodies were used for staining of vaginal dendritic cells: Fixable Aqua Dead Cell Stain Kit (Thermo Fisher), CD45 (clone 30-F11, BioLegend), CD11b (clone M1/70, BioLegend), and CD11c (clone N418, BioLegend). All cells were stained in 1% BSA PBS and incubated on ice for 15 to 20 minutes. Cells were acquired on BD LSRII cytometer and analyzed by FlowJo software v8.8.7 (Tree Star, Inc.).
9
Multiparameter Flow Cytometry Panel
10
Tumor Cell and Immune Cell Profiling
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As described previously35 , subcutaneous tumours with KP1.9 cells were harvested from C57BL/6 flanks 3 weeks after implantation, minced, and shaken at 600 r.p.m. with 0.2 mg ml−1 collagenase type I (Worthington Biochemical Corporation) in RPMI-1640 for 30 min at 37 °C. Digested samples were filtered (70 μm BD Falcon strainer); washed in PBS with 0.5% BSA and 2 mM EDTA; incubated with Fc-block (TruStain fcX anti-mouse CD16/32; clone 93; Biolegend) for 15 min at 4 °C; and labelled with antibodies as indicated for 45 min at 4 °C. Flow cytometry (LSRII, BD Biosciences) labelled tumour cells (CD45− EpCAM+), TAM (CD45+ CD11b+ Ly6C- Lin- CD11c+ F4/80+), lymphocyte-like cells (CD45+ CD11b- Lin+), along with CD45- EpCAM- host-cell populations. Antibodies included EpCAM (clone G8.8; eBioscience); CD45 (clone 30-F11; Biolegend), F4/80 (clone BM8; Biolegend), CD11c (clone N418; Biolegend), Ly6C (clone HK1.4; Biolegend); and CD11b (clone M1/70; BD Biosciences). The lineage (Lin) antibody mix contained anti-CD90.2 (clone 53–2.1), anti-B220 (clone RA3-6B2), anti-NK1.1 (clone PK136), anti-CD49b (clone DX5), anti-Ter119 (cloneTER-119) and anti-Ly6G (clone 1A8) (all BD Biosciences). 7-aminoactinomycin D (7-AAD, Sigma Aldrich) excluded dead cells. VT680 fluorescence was directly assessed using the LSRII flow cytometer, FlowJo v.8.8.7 (Tree Star, Inc.) and MATLAB.
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