Differentiated AST-OPC1 samples were assayed for the presence of surface and intracellular markers using standard flow cytometry. For surface marker staining, day 41 AST-OPC1 samples were blocked and incubated with primary antibody and/or isotype control (NG2, cat # 37-2300, Invitrogen, CA, USA; Mouse IgG1, cat # 554121, BD Biosciences, CA, USA) followed by fluorescent-conjugated secondary antibodies. For intracellular marker staining, day 41 AST-OPC1 samples were tagged with ethidium monoazide (cat # E2028, Sigma, MO, USA) for dead cell discrimination, followed by fixation using 2% paraformaldehyde (PFA) and then permeabilization with cold 90% methanol. The cells were blocked, and then incubated with primary antibody and/or isotype control (Oct4, cat # SC8629, Santa Cruz, CA, USA; Goat IgG, cat # SC2028; Santa Cruz; Nestin, cat # MAB5326, Millipore, MA, USA; Mouse IgG1, cat # 554121, BD Biosciences) followed by fluorescent-conjugated secondary antibodies. All samples were then acquired and the data analyzed on the BD Biosciences FACSCalibur™ cytometer system using Cellquest Pro software (BD Biosciences).
Mouse igg1
Manufactured by BD
Sourced in United States
Mouse IgG1 is a type of immunoglobulin G (IgG) antibody isolated from mouse serum. IgG1 is the most abundant IgG subclass and plays a crucial role in the adaptive immune response. It is commonly used in research and diagnostic applications as a reference standard or control.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg2a, by BD (4 mentions)
Cellquest pro software, by BD (3 mentions)
Igg2a, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Igg2b, by BD (2 mentions)
Fitc conjugated goat anti mouse igg, by BD (2 mentions)
Cellquest software, by BD (2 mentions)
Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (2 mentions)
Ne per extraction kit, by Thermo Fisher Scientific (2 mentions)
Gel shift kit, by Panomics (2 mentions)
Anti stat3 antibodies, by BD (2 mentions)
Cd11b, by BD (2 mentions)
Mouse igg1, by R&D Systems (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ethidium monoazide, by Merck Group (1 mentions)
Mouse igg1, by Fortrea (1 mentions)
Fortessa x20, by BD (1 mentions)
Goat anti mouse igg pe, by Abcam (1 mentions)
Rat anti mouse cd31 antibody, by BD (1 mentions)
Anti rat igg dynabeads, by Thermo Fisher Scientific (1 mentions)
48 protocols using mouse igg1
1
Flow Cytometric Analysis of AST-OPC1 Cells
2
Immunohistochemical Labeling of Frozen Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slides carrying frozen tissue sections were thawed at room temperature and post-fixed in 4% paraformaldehyde for 10 min. High-protein blocking solutions (5% of goat or donkey serum or 2% bovine serum albumen, in PBS) were used to reduce non-specific antibody binding. The primary antibodies were diluted in blocking solution as follows: anti-GFP (1:1000, rabbit IgG, Invitrogen, Waltham, MA, USA, A11122), HA.11 (1:500, mouse IgG1, Covance, Princeton, NJ, USA, MMS-101P), dsRNA (1:500, J2, mouse IgG2a, SCICONS), 3C2 (1:5–1:40, mouse IgG1 hybridoma culture supernatant) [63 (link)], anti-HCS-1 (1:100, mouse IgG, BD Biosciences, San Jose, CA, USA), and axonal marker 3A10 (1:50, mouse IgG1, DSHB #3A10, AB_531874). Fluorophore-tagged secondary antibodies (Life Technologies, Carlsbad, CA, USA) were used at 1:250 or 1:500 dilution for fluorescence microscopy. For 3C2 and HCS-1/3A10 immunohistochemistry, diaminobenzidine (DAB) calorimetric staining was performed with a biotin-tagged secondary antibody and the ABC avidin–biotin–HRP enhancement step (Vector Laboratories, Newark, CA, USA), followed by diaminobenzidine oxidation reaction.
3
Phenotypic Characterization of Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cMSCs and pbMSCs were defined according to the 3 criteria of the International Society for Cellular Therapy [13 (link)]: (1) adhesion to plastic, (2) expression of a specific combination of surface markers, and (3) differentiation potential (trilineage differentiation into adipocytes, osteoblasts, chondrocytes, and blood vascular cells) [10 (link)]. To define the phenotype of cMSCs and pbMSCs, we analyzed cultured cells at passage 4 for rat MSC markers by flow cytometry using the following fluorescence antibodies: SH2, SH3, CD90, CD147, CD34, CD45, and CD133. Mouse IgG1, IgG2a, and IgG2b (Becton Dickinson) were used as isotype controls, and marker expression was evaluated using FACS.
4
FPR Surface Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FPR surface expression analysis, 2 x 105 PHK or N/TERT-1 cells were incubated in a 96-well V-bottom plate either with the isotype control (mouse IgG1, Becton Dickinson), anti-FPR1 mouse anti-human FPR1 (BD Bioscience) or anti-FPR2, each 5 µl (mouse anti-human FPRL1, Alvedron Freiburg) for 40 min on ice in PBS, after two washing steps, each time using 250 µl PBS. Cells were stained with a second fluorescently labelled antibody (goat anti mouse IgG-PE, Abcam) for 25 min on ice in the dark. After two washing steps, cells were analyzed by flowcytometry (Fortessa X-20, Becton Dickinson) and FlowJo™ software.
5
Isolation of Brain Endothelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain endothelial cells were isolated using a modification of a published protocol (Lim and Luscinskas, 2006) . Mouse IgG1 (349040; BD) was added to deplete FcR-expressing cells (1 mg/mL). Rat anti-mouse CD31 antibody (553370; BD PharMingen) coated sheep anti-rat IgG Dynabeads (11035; Invitrogen) were then used to bind CD31-positive endothelial cells and were separated using a magnetic separator.
6
Characterization of hDPSCs Surface Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expressions of cell surface markers for hematopoietic and MSC marker proteins were analyzed using flow cytometry as previously described 15 . Briefly, hDPSCs-fresh and hDPSCs-cryo were harvested using 0.25% trypsin-EDTA and fixed in 3.7% formaldehyde solution. The cells were washed twice with DPBS and labeled with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD34 (BD Pharmingen, San Jose, CA, USA), FITC mouse anti-human CD45 (Santa Cruz Biotechnology, Dallas, TX, USA), FITC mouse anti-human CD90 (BD Pharmingen), unconjugated mouse monoclonal CD73 (Santa Cruz Biotechnology), and unconjugated mouse monoclonal IgG2a (CD105; Santa Cruz Biotechnology,) for 30 min. Unconjugated primary antibodies were treated with secondary FITC-conjugated goat anti-mouse IgG (BD Pharmingen) for 30 min in the dark. The isotype control was mouse IgG1 (BD Pharmingen). A total of 15,000 labeled cells per sample were acquired. The results were analyzed using cell Quest Pro software (Becton Dickinson) (Fig. 2 ).
7
Evaluating Cytotoxic Function via CD107 Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CD107 externalization assay was performed to evaluate cytotoxic function.32 (link) Cells (2×105) were cocultured with the same number of Daudi cells in the presence of 10 μmol/L Monensin (Sigma) and 5 μL of FITC-conjugated anti-CD107a and CD107b antibody (BD) or isotype control (mouse IgG1, BD). After 4 hours incubation, CD107 expression was determined by flow cytometry as an indicator of degranulation of cytotoxic molecules. At the same time, culture supernatants were harvested and analyzed for cytokine and chemokine production using Bio-Plex Pro Human Cytokine Grp I panel 27-plex (BIO-RAD, Hercules, California, USA).
8
Integrin regulation of MMP expression
9
Characterizing DC Surface Phenotypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To characterize the cell surface phenotypes on DCs, flow cytometry was performed using a FACSAria cell sorter (Becton Dickinson, San Jose, CA, USA) after labeling of the cells with CD86-PE, CD83-FITC, CCR7-FITC (PharMingen, San Diego, CA, USA), and the relevant isotype controls (mouse IgG1 and IgG2a, PharMingen). Cell debris was eliminated from the analysis by forward and side-scatter gating, and the data were analyzed with WinMDI Version 2.9 software (Biology Software Net).
10
Immunohistochemical Analysis of Formalin-Fixed Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed 4 μm sections were deparaffinised with xylene and rehydrated through graded ethanol and demineralised water. Antigens were retrieved by boiling in 10 mM citrate buffer, pH 6.0 in a pressure cooker. Non-specific binding was blocked using Antibody Diluent S2022 (Dako). Consecutive tissue sections were incubated with the following primary monoclonal mouse anti-human antibodies diluted in Antibody Diluent S2022 (Dako): anti-TF (No. 4509, American Diagnostica), anti-Epithelial Membrane Antigen (MUC1) (clone E29), anti-CD68 (clone KP1) and anti-CD31 (Clone JC70A) from Dako. After blocking endogenous peroxidases, staining was visualised using a peroxidase-labelled polymer conjugated to goat anti-mouse immunoglobulins and 3,3′-diaminobenzidine (K4007, Mouse EnVision+ kit, Dako). Sections were decolourised, counterstained with haematoxylin (1 : 1 dilution with demineralised water; Merck, Darmstadt, Germany), decolourised again, dehydrated and finally permanently mounted.
Mouse IgG1 (BD Biosciences) and omission of the primary antibody served as specificity controls. Normal pancreatic islet cells, tonsils and ductal breast cancer cells served as positive control for TF, CD68 and MUC1 staining, respectively. No separate control was prepared for CD31 staining.
Mouse IgG1 (BD Biosciences) and omission of the primary antibody served as specificity controls. Normal pancreatic islet cells, tonsils and ductal breast cancer cells served as positive control for TF, CD68 and MUC1 staining, respectively. No separate control was prepared for CD31 staining.
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!