Proteins were separated by SDS-PAGE before being subjected to Western blot. The following antibodies were from Santa Cruz: p24 IF antibody (sc69728), Myc (9E10), DDX17 N Terminus (sc-398168) and DDX17 (sc-271112); Abcam: SRSF1/SF2 (ab129108), Tetherin/BST2 (ab14694), Vpu (ab134061), U2AF65 (ab37530), rabbit isotype-matched control IgG (ab27478), rabbit c-Myc (ab39688), Nef (ab42355), Rev (ab855299) and GAPDH (ab9485). Vif antibody was from GeneTex (GTX80393). U1C antibody was obtained from Bethyl Laboratories (A303-947A). DDX5 antibody PAb-204 was a kind gift from Dr. Frances Fuller-Pace (Dundee). HIV-1 p55/p24 (ARP, NIBSC) [67] (link). Envelope/gp120 Mouse Monoclonal was obtained from CFAR. Mouse IgG isotype control (401402) was obtained from Biolegend. The following secondary antibodies were from Cell Signalling: horseradish peroxidase-conjugated anti-mouse (#7076, 1:2000), and from Santa Cruz: horseradish peroxidase-conjugated anti-rabbit (#2123, 1:2000). Detection was carried out using ECL prime (Amersham) per the manufacturer’s instructions.
Mouse igg isotype control
Manufactured by BioLegend
Sourced in Japan, United States
The Mouse IgG isotype control is a laboratory reagent used to establish background staining levels in flow cytometry and other immunoassays. It serves as a negative control to help distinguish specific from non-specific antibody binding.
Automatically generated - may contain errors
Lab products found in correlation
Ddx17, by Abcam (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Nis elements ar imaging software, by Nikon (1 mentions)
Eclipse te2000 e inverted fluorescence microscope, by Nikon (1 mentions)
Anti cd18, by BioLegend (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Anti cd62l, by BioLegend (1 mentions)
Alexa fluor 488 goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Anti cd31, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 h342, by Dojindo Laboratories (1 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Facscanto 2, by BD (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
Cytofix cytoperm kit with golgistop, by BD (1 mentions)
Diva software, by BD (1 mentions)
Facsaria, by BD (1 mentions)
6 protocols using mouse igg isotype control
1
Western Blot Analysis of HIV-1 Proteins
2
Assessing Apoptosis-Related Protein BAX
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were trypsinized and then fixed in 2% formaldehyde for 10 min at room temperature. Washed cells were resuspended in PBS. Anti-BAX antibody (BD Biosciences clone 6A7, 1 μg) was added to 100 μl of permeabilization buffer (0.1% saponin, 0.5% BSA in PBS), in which cells were incubated for 1 h at 4 °C. Mouse IgG isotype control (Biolegend) served as an autofluorescence control. Samples were washed and incubated with a 1:200 dilution of FITC-conjugated anti-mouse antibodies in PBS for 1 h at 4 °C. Samples were washed and resuspended in 300 μl PBS and analyzed by flow cytometry. Histograms were overlaid using FCS express.
3
Divalent Cation Effects on Cell Aggregation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Disaggregated cells were seeded at a density of 1 × 105 cells/ml in 8 mL of complete RPMI 1640 medium and the formation of aggregates was examined at 72 hours of culture by phase contrast with a 10 × objective. To examine the effect of divalent cations on aggregate formation, cells were cultured in the presence or absence of 1 mM EDTA (Sigma-Aldrich) or EGTA (Sigma-Aldrich) for 24 h. For antibody-blocking experiments, cells were placed in 96-well culture plates at a density of 0.5–1 × 106 cells/ml in 100–150 μl/well of complete RPMI 1640 medium containing 10 µg/ml anti-CD18 (Biolegend), anti-CD62L (BioLegend) mAbs or mouse IgG isotype control (BioLegend) and then incubated at 37°C for 2 h. Photomicrographs were taken using an ORCA-ER monochrome cooled-CCD camera (Hamamatsu, Hamamatsu, Japan) coupled to an Eclipse TE2000-E inverted fluorescence microscope (Nikon, Tokyo, Japan) and NIS-Elements AR imaging software and the number of aggregates containing more than 20 cells per field was counted.
4
Immunocytochemistry and Flow Cytometry for CD31
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunocytochemical analysis, we fixed the cells with 4% paraformaldehyde for 10 min, and permeabilized it with a PBS solution containing 0.1% of Triton-X 100 for 10 min. After blocking with 5% FBS, we incubated the cells with anti-CD31 (1/30; 14-0319-82, Thermo Fisher Scientific, Waltham, MA, USA) primary antibody at 4°C overnight. Afterward, we incubated the cells with secondary Goat anti-Mouse Alexa Fluor 488 antibody (1/400; A-11001, Thermo Fisher Scientific) for 60 min at room temperature, and we stained the nuclei with Hoechst 33 342 (H342, Dojindo Molecular Technologies, Kumamoto, Japan). We developed the images by IN Cell Analyzer 6000 (GE Healthcare, Chicago, IL, USA). For flow cytometry, we suspended the cells in HBSS containing 3% FBS and 0.3 mM EDTA, fixed with 4% paraformaldehyde, and stained with the same antibodies used in immunofluorescence. Mouse IgG isotype control (401 401, BioLegend, San Diego, CA, USA) was applied as a negative control. We analyzed the cells via a BD FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA), and performed the data analysis using FlowJo v10 software (BD Biosciences).
5
Detecting Intracellular Cytokines in Human PBMC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometric detection of intracellular IL-17 and IFNγ in human PBMC was carried out after an extended period of culture as described above. After 13–14 days of culture in the absence or presence of R and corresponding Cit peptides, a staining protocol and a fixation/permeabilization kit (Cytofix/Cytoperm kit with GolgiStop) from BD Biosciences were employed to detect intracellular cytokines as described previously [32 (link)]. In brief, the cells (2 x 106/ml culture medium) were first incubated with 10 ng/ml phorbol-13-myristate acetate (PMA, Sigma-Aldrich), 1 μg/ml ionomycin (Invitrogen, Grand Island, NY, USA), and 1 μl/ml GolgiStop (containing 2 μM monensin) for 4 hours. After blocking Fc receptors with a mouse anti-human CD16/32 mAb, followed by surface staining with a fluorochrome-conjugated mouse mAb against human CD4 (BioLegend, San Diego, CA, USA), the cells were fixed, permeabilized, and stained with fluorochrome-conjugated mouse mAbs to human IL-17A and IFNγ (BioLegend). Flow cytometry was performed using a BD FACS Canto II instrument, and data were analyzed with FACS Diva software (BD Flow Cytometry Systems, San Jose, CA, USA). Non-specific background fluorescence was determined by staining a separate aliquot of human cells with mouse IgG isotype controls (BioLegend) matching the isotypes and fluorochromes of the CD4- and cytokine-specific mAbs.
6
Quantifying CD45 Subsets by Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Determination of cell surface expression of CD45RABC and CD45RO molecules was carried out by cytofluorimetric analysis using the FACS ARIA cell-sorting system and DIVA software (BD Biosciences). Direct immunofluorescence was performed using PerCP and FITC mouse anti-human CD45RABC and CD45RO antibodies respectively, along with the appropriate mouse IgG isotype controls (BioLegend). Staining, washing and analysis were performed following the manufacturer's recommendations.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!