Colon crypt single cell suspensions were DNAse treated for 5 min (Sigma-Aldrich #4716728001). Following a wash step, cells were incubated for 30 min in FACS buffer (phosphate buffered saline with 3% fetal bovine serum + 10 µM Rock inhibitor (Y-27632 AdipoGen Life Sciences from Fisher #501146540)) with the following pre-conjugated validated flow antibodies: CD45-BV510 (1:200, Clone 30-F11; BD Biosciences #563891), CD31-BV510 (1:200, Clone MEC 13.3; BD Biosciences #563089), CD326-eFluor450 (1:100, Clone G8.8; eBioscience #48-5791-82), CD44-PerCP-Cy5.5 (1:100, Clone IM7; Thermo Fisher #A26013), CD24-PECy7 (1:200, Clone M1/69; eBioscience #25-0242-82), and CD117-APC-Cy7 (1:100, Clone 2B8; Thermo Fisher #A15423). Following wash steps, cells were resuspended in FACS buffer and Live/Dead Aqua (Thermo Fisher # L34957). An alternative CD45-APC (1:200, Clone 30-F11; BD Biosciences #561018) antibody was used in the Supplementary Fig. 7f where specified.
Clone 30 f11
Manufactured by BD
Sourced in United States
The Clone 30-F11 is a laboratory instrument designed for cell culture applications. It is capable of performing cell counting and viability analysis. The device utilizes automated methods to provide precise and consistent measurements of cell samples.
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Lab products found in correlation
Clone m1 70, by BD (3 mentions)
Clone 53 6, by BD (3 mentions)
Cytofix cytoperm plus fixation permeabilization kit with golgiplug protein transport inhibitor containing brefeldin a, by BD (2 mentions)
Clone gk1, by BD (2 mentions)
Clone xmg1, by BD (2 mentions)
Dispase, by BD (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Dispase, by Merck Group (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Clone mec 13, by BD (1 mentions)
Clone m1 69, by Thermo Fisher Scientific (1 mentions)
Clone g8, by Thermo Fisher Scientific (1 mentions)
Zombie nir, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsymphony flow cytometer, by BD (1 mentions)
Versacomp antibody capture bead kit, by Beckman Coulter (1 mentions)
Streptavidin pe, by BD (1 mentions)
Anti cd31 pe, by BD (1 mentions)
Anti cd45 af700, by BD (1 mentions)
Clone 11d4, by BD (1 mentions)
22 protocols using clone 30 f11
1
Colon Crypt Single Cell Isolation
2
Multiparameter Flow Cytometry of Immune Cells
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Single cell suspensions were incubated with primary antibodies against CD11b (1:400, clone M1/70, BD Biosciences, 565976), CD45 (1:800, clone 30-F11, BD Biosciences, 563890) or CD45 (1:400, clone 30-F11, BD Biosciences, 565079), CD22 (1:100, clone OX-97, Biolegend, 126112) as well as live cell identification using Zombie NIR™ (1:500, Biolegend, 423106) in PBS for 30 min at 4°C. After washing once with PBS, samples were resuspended in 200 µl PBS and analyzed by flow cytometry with a BD LSRFortessa (BD Biosciences) or a BD FACSymphony flow Cytometer (BD Biosciences). To reduce fluorescent spillover, PMT voltages were adjusted with single-stain controls prepared with VersaComp Antibody Capture Bead Kit (Beckman Coulter, B22804) before sample acquisition. Acquired.fcs files were uploaded in FlowJo software (Tree Star), where artefacts arisen from incorrect compensation-matrix calculation were manually corrected.
3
Immunostaining Tumor-infiltrating Lymphocytes
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Tissues e.g. tumors, skin and lymph nodes were harvested when mice were killed. Tissue specimens were immersed in a zinc-based fixative (DAKO), embedded in paraffin, and routinely stained with hematoxylin and eosin. To confirm the melanocytic origin of tumor cells infiltrating the draining lymph node, sections were analyzed by an immunofluorescence double staining using the Trp1-specific polyclonal rabbit antibody (PEP7, a kind gift from Vincent Hearing, NIH, Bethesda) and a monoclonal rat anti-CD45 antibody (Clone 30-F11, BD Bioscience) followed by appropriated enzyme-conjungated secondary antibodies and the LSAB-2 color development system 8 (DAKO) or by fluorochrome-conjugated secondary antibodies, anti-rabbit Alexa488 and anti-rat Alexa594 (Jackson Immunoresearch). Heavily pigmented lymph nodes were bleached by incubation in 3% H2O2 and 0.5% KOH at 37°C for 20 min. Slides were placed into 1% acetic acid for 20 sec and washed in TRIS buffer for 5 min. Antigen-retrieval was done by incubation in citrate buffer pH = 6,0 and steaming for 20 min. CD45+ immune cells were counted in three sequential high-power-fields (20× magnification) and mean number of immune cells were expressed as CD45+ immune cells/3 high-power fields. Stained sections were examined with a Leica DMLB immunoflourescence microscope and images were acquired with a JVC digital camera KY-75FU.
4
Characterization of AGM Hematopoietic Cells
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Single-cell suspensions from the AGM region were prepared by dispase/collagenase-mediated dissociation. Antibodies used for staining of cells were anti-CD45-BV450 (BD Horizon, clone 30F11, 1:100), anti-CD45-AF700 (1:100, clone 30F11, BD pharmingen), anti-VE-cadherin-AF647 or -AF488 (1:100, Clone eBioBV13, Biolegend) and biotinylated anti-VE-Cadherin (1:50, clone 11.D4.1, Pharmingen), followed by incubation with streptavidin-PE (1:600, BD Pharmingen), anti-CD43-PE (1:200, clone eBioR2/60, eBioscience), anti-cKit-APC (1:100, clone 2B8, eBioscience), anti-CD31-PE (1:200, MEC13.3, Pharmingen), anti-Sca1-V500 (1:100, clone D7, BD Bioscience), anti-CD41-BV421 (1:100, clone MWreg30, Biolegend) and anti-Ter119-PerCp-Cy5.5 (1:100, clone TER119, eBioscience). 7-Aminoactinomycin D viability staining solution was used to exclude dead cells, and gates were set using appropriate fluorescence minus one controls. Sorting was performed on a FACSAriaII using the FACSDiva software.
5
Immunophenotyping of Tumor-Infiltrating Leukocytes
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Transplanted melanoma infiltrating immune cells were isolated as described previously and stained with fluorochrome-conjugated monoclonal antibody specific for mouse CD45 (clone 30-F11, 1:200), CD11b (clone M1/70, 1:200), Gr1 (clone RB6–8C5, 1:200), CD8a (clone 53-6.7, 1:200) and CD4 (clone GK1.5, 1:200; all from BD Pharmingen) according to standard protocols. Data were acquired with a FACSCanto Flow Cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar, V7.6.5 for Windows).
6
Characterizing HER3-specific T cells
7
Isolation of Mouse Microglia by FACS
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Cells from young, middle-aged and aged mice were sorted by FACS as previously described41 (link),45 (link). In brief, the mixed brain cells were resuspended in FACS buffer. Then, mixed cells were stained with antibodies against CD11b (1:100, clone M1/70, BD Pharmingen, 557657) and CD45 (1:100, clone 30-F11, BD Pharmingen, 553080) in FACS buffer for 30 min on ice (Supplementary Table 8 ). Dead cells were labeled with 7-AAD (1:80, BD Pharmingen, 559925). Then, CD11b+ CD45low 7-AAD− microglia were collected by FACSAria III cell sorting (BD Biosciences). For brain cell scRNA-seq (except for 3xDR versus control groups), 7-AAD− brain cells were collected. Harvested cells were then used for scRNA-seq, bulk RNA-seq and ATAC-seq.
8
Quantifying Human Cell Engraftment
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Harvested cells were stained with antibodies against mouse (clone 30-F11, BD Pharmingen, San Jose, CA, USA) and human (clone HI30, BD Pharmingen, San Jose, CA, USA) CD45. Flow cytometry was performed on an LSR II (BD Biosciences, San Jose, CA, USA) and data analyzed using FlowJo software (TreeStar, Ashland, OR). CG-SH engraftment levels were determined by quantifying the percentage of human CD45 positive (hCD45+) cells by flow cytometry.
9
Immunohistochemical Analysis of Oxidative Stress Markers
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For immunohistochemistry analyses, 10 mice were sacrificed at five different time points post-MVD induction (n=2 per time point at 3 h, 24 h, 3 d, 7 d, and 14 d), and an additional 2 mice with sham surgery were also assessed to match previously described histological analyses. Frozen sections (6 μm thick) were cut with a cryostat, mounted on glass slides, and fixed for 10 min with acetone at -20 °C. Sections were pre-incubated with 10% normal goat serum and / or bovine serum albumin (BSA) for 15 min to block nonspecific bindings of antibodies. These sections were incubated with either a monoclonal antibody to 4-hydroxy-2-nonenal (4-HNE; Oxis; diluted 1:10) or a polyclonal antibody to nitrotyrosine (Sigma; 1:500); or mouse anti-mouse monoclonal CD68 antibody (ab955) (1:200 dilution; clone KP-1; ABCAM), rat anti-mouse CD45 (1:200 dilution; clone 30F-11; BD Biosciences — Pharmingen), or nonimmune rat IgG (1:100 dilution; DakoCytomation A/S) overnight at 4 °C and the secondary antibody (fluorescent anti-mouse or anti-rabbit IgG reagent 1:500 dilution; Invitrogen) for 1 h at room temperature. The negative control was processed in a similar fashion by replacing the first antibodies with nonimmune (rat) IgG. The fluorescent reaction product was visualized utilizing 4-laser confocal microscope.
10
Isolation and Culture of Alveolar Epithelial Cells
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Lung homogenates were obtained by instillation of dispase (BD Biosciences) through the trachea into HBSS (Gibco) perfused lungs, followed by incubation (in dispase) for 40 min as previously described61 (link). After removal of the trachea and proximal bronchial tree, the lungs were homogenized (GentleMACS, MACS Miltenyi Biotech) in DMEM/2.5% HEPES with 0.01% DNase (Serva) and filtered through 100 and 40 μm nylon filters. Cell suspensions were incubated with biotinylated rat anti-mouse CD45 (Clone 30-F11, BD, Cat No 553078), CD16/32 (clone 2.4G2, BD, Cat No 553143) and CD31 (clone MEC13.3, BD, Cat No 553371) mAbs for 30 min at 37 °C followed by incubation with biotin-binding magnetic beads and magnetic separation to deplete leukocytes and endothelial cells prior to further culture. AEC suspensions with a purity ≥90% as determined by FACS were seeded at a density of 120–150,000 cells/cm2 in 4-μm–pore size transwells (Corning Inc.), in 24-well plates at a density of 250,000 cells/cm2, or in chamber slides (Corning Inc.), and cultured in DMEM enriched with HEPES, L-Glutamine, FCS, and pen/strep. The list of antibodies in Excel format including clone, company name, Cat No is provided as Supplementary Data 1 .
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