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9 protocols using isotype control

1

Antibody Pretreatment of Hepatocytes

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Primary human hepatocytes or compacted hepatocyte-fibroblast spheroids (immediately after harvest from microwell molds) were incubated with 10 μg/mL functional blocking monoclonal antibody (mouse anti-human β1 integrin, clone P5D2; mouse anti-human E-cadherin, clone 67A4; EMD Millipore) or an isotype control (Santa Cruz Biotechnology) for 20 minutes at 37 °C in hepatocyte media. Excess antibody was removed by centrifugation at 60×g for 6 minutes for 3 washes total. Pelleted hepatocytes were then encapsulated in fibrin hydrogels and cultured in hepatocyte media.
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2

Quantifying DNA Damage in Drug-Treated Cells

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Experiments for confocal imaging were plated on poly-d-lysine-coated chamber slides (Corning; 354632). Cells were treated with DMSO, AZD1775 IC15, gemcitabine IC15, or the drugs in combination, and fixed in 4% paraformaldehyde after 48 hours. Cells were permeabilized in 0.2% Triton-X and then blocked in 5% milk in 0.05% Triton-X. Primary antibodies were p-γH2AX (Cell Signaling; 9718) or isotype control (Santa Cruz Biotechnology; sc-2027). Secondary antibody was conjugated with Alexa Fluor 488 (green). Nuclei were stained using DAPI (ProLong Gold antifade reagent with DAPI, Life Technologies). Images were captured using a 20× objective for quantification and 40x oil objective for representative images using a 3I Marianas inverted spinning disk confocal microscope and Evolve 16-bit EMCCD camera. Quantification was performed using Image J by normalizing p-γH2AX intensity to the DAPI signal.
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3

FACS Analysis of Cell Surface Markers

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For FACS analysis adherent cells at about 70% confluence were detached using 0.1 mM EDTA in PBS (Sigma Aldrich, Darmstads, Germany), centrifuged and re-suspended in PBS containing 0.2% BSA. Cell aliquots (2.5 × 105 cells) were treated with primary monoclonal antibody PE conjugate, (Millipore, Burlington, MA, USA) or isotype control (Santa Cruz Biotechnology, Dallas, TX, USA), at the same concentration (14 µg/mL), whole a reaction volume of 50 µL for 30 min at 4 °C. After washing, the cells were analyzed by using a flow cytometer equipped with a 488 nm argon laser (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA). A total of 20,000 events per sample were collected. Integrin quantification with Quantibrite PE beads (BectonDickinson Biosciences) was evaluated as previously described [46 (link)]. Values of fluorescence intensity were obtained from the histogram statistic of CellQuest software.
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4

Quantitative Analysis of Cyclin Expression

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Staining for cyclins was performed according to the method described by Darzynkiewicz et al38 (link). Briefly, subconfluent cultures of T98G and MO59K cells were treated with the indicated concentrations karenitecin in the presence and absence of radiation for 72 hours. The cells were harvested and fixed in ice-cold ethanol overnight at 4° C. The cells were washed, centrifuged, and suspended in permeabilization solution (0.25%v/v Triton X-100 in PBS pH 7.4) and kept on ice for 5 minutes. The cells were washed again, resuspended in 100μl wash buffer (1% BSA in PBS); and incubated with antibodies to cyclins B, D or isotype control (Santa Cruz biotechnology, CA) for 60 minutes. The cells were washed, and stained with goat anti-mouse FITC for 30 minutes at RT. The stained cell preparation was stained with 5 μg/ml PI and 200 μg/ml DNase-free RNase A. The cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and the results were acquired using CELL quest software and the Modfit LT version 2.0 (Verity Software, Topham, Maine). PI staining was used to reveal the various phases of the cell cycle and the cyclin staining in the G0/G1 and G2/M phase of the cell cycle was estimated as the MFI after subtracting the Mean Fluorescence Intensity recorded by the isotype control for each sample.
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5

LSEC CD36 Antibody Binding Assay

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CTR LSECs were plated on fibronectin-coated in 10% FBS-DMEM, at 37°C/5%CO2. Non-adherent cells were removed and LSECs were incubated for 15 min with FBS-free DMEM, anti-human CD36 Antibody (0.01μg/mL - STEMCELL™ Technologies, cat#60084) or Isotype control (0.01μg/mL - Santa Cruz Biotechnology, Cat# sc-3877). Then, cells were and harvested for cGMP measurement.
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6

Flow Cytometric Analysis of Integrin Expression

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Adherent cells at about 70% confluence were detached using 0.1 mM EDTA in PBS (Sigma Aldrich), centrifuged and suspended in PBS containing 0.2% BSA. Cell aliquots (2.5×105 cells) were treated with primary monoclonal antibody, clone LM609 (Millipore MA, USA) or isotype control (Santa Cruz Biotechnology, Germany), at the same concentrations for 30 min at 4°C. After washing, the cells were incubated with secondary antibody FITC-conjugated (Santa Cruz Biotechnology, Germany), washed and analysed by using a flow cytometer equipped with a 488 nm argon laser (FACScan, Becton Dickinson, USA). A total of 20000 events per sample were collected. Values of fluorescence intensity were obtained from the histogram statistic of CellQuest software.
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7

Isolation and Characterization of Immune Cells

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Ficoll -Hypaque solution (Lymphoprep; Nycomed Pharma, Oslo, Norway), RPMI 1640 medium and phosphate buff ered saline (PBS) were purchased from HyClone Laboratories Inc. (Logan, UT). Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Grand Island, NY). Fluorescent-conjugated monoclonal antibodies directed to human CDs were purchased from BD Biosciences (San Jose, CA). Goat anti-human high-mobility group nucleosome binding protein 2 (HMGN2) antibody and isotype control were from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Th e Daudi cell line was obtained from the American Type Culture Collection (ATCC).
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8

Neutrophil Surface Marker Profiling

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Antibody staining was carried out on freshly isolated neutrophils incubated for up to 1 h, as described above. Neutrophils (1 × 105) were resuspended in PBS (+ 0.2% BSA). Antibody binding was carried out at 4 °C in the dark for 30 min with conjugated antibodies added as follows: CD62L-FITC (R&D systems); CD11b-PE (R&D systems); CD16-PE (R&D systems); CD18-PE (R&D systems); CD63-APC (Thermo-Fisher); CD64-FITC (R&D systems); IL-8R (CXCR1)-FITC (R&D systems); CD66b-FITC (R&D systems); and isotype controls (Santa Cruz). Fluorescence was measured immediately on a Dako Cyan ADP flow cytometer. 10,000 events/sample were analysed.
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9

Immune Cell Profiling of Mouse Skin

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Mouse dorsal skins were formalin-fixed and paraffin-embedded. Hematoxylin and eosin (H&E) staining of paraffin-embedded section (4–5 μm) was conducted according to standard methods. Immunohistochemical staining was performed following the regular procedure with the following antibodies: anti-CD3 (sc-20047), anti-CD4 (sc-7219) and anti-CD8 (sc-7188). Isotype controls (Santa Cruz) were included in each staining. Slides were examined using an Olympus microscope. Quantification of H&E and IHC staining was performed by two experimenters for two sections of two mice per treatment group. Numbers of positive cells were counted for five high power fields (HPFs) and averaged. Immunofluorescent staining was performed following the regular procedure with the following antibodies: anti-CD3 (145-2C11), anti-CD4 (RM4-5), and anti-CD25 (PC61) antibodies (BD Biosciences, San Jose, CA, USA).
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