The ICAM1 blocking assay was performed using 1, 5 or 25 μg/ml mouse anti-ICAM1 antibody (ab2213; Abcam, Japan). An isotype control antibody (25 μg/ml) was used as control. Cells were seeded onto a 12-well plate for 3 days and then treated with a serial dilution series of anti-ICAM1 antibody for 1 h at 37 °C. The cells were tested for PM adherence.
Ab2213
Manufactured by Abcam
Sourced in United Kingdom, United States, Japan
Ab2213 is a recombinant monoclonal antibody targeting an undisclosed antigen. It is intended for use as a research tool.
Automatically generated - may contain errors
Lab products found in correlation
Ab134047, by Abcam (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab6672, by Abcam (2 mentions)
Ab32441, by Abcam (1 mentions)
Sc15404, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab126250, by Abcam (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab52771, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Detergent compatible bradford protein quantification kit, by Vazyme (1 mentions)
Pvdf membranes, by Vazyme (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Ripa buffer, by Vazyme (1 mentions)
Ecl kit, by Vazyme (1 mentions)
Draq5, by Thermo Fisher Scientific (1 mentions)
A22287, by Thermo Fisher Scientific (1 mentions)
Ab150117, by Abcam (1 mentions)
16 protocols using ab2213
1
ICAM1 Blocking Assay Protocol
2
Immunoblotting Analysis of Cellular Signaling
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Whole cell lysates were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Western blotting. We used antibodies against USP18 (sc-374064), AKT (sc-8312), phospho-AKT (sc-7985R), phosphotyrosine (sc-7020) from Santa Cruz at dilution 1/500; against NFκB p65/RelA (8242S), phospho(S536)-NFκB p65/RelA (3033S), insulin receptor beta-subunit (3025), JNK (9252), phospho-JNK (9251S), TAK1 (45055) and phospho-TAK-1(4531) from Cell Signalling Technology, Inc. (Danvers, MA, USA) at dilution 1/1000; against p16 (10883-1-AP) and p21 (10355-1-AP) from Proteintech (Proteintech Europe, Manchester, UK) or from BD Biosciences (San Jose, CA, USA) at dilution 1/1000; against ICAM-1 (ab2213) and VCAM-1 (ab134047) from Abcam (Abcam, Cambridge, UK) at dilution 1/1000; against tubulin, used as an index of the cellular protein content, from Sigma-Aldrich (Saint Louis, MO, USA) at dilution 1/1000. For antibodies against SIRT1 we used at first antibodies from Santa Cruz (sc15404) at dilution 1/500 then from Abcam (ab32441) at dilution 1/1000.
The activity of NFκB was measured by the ratio of the level of phosphorylation of the p65/RelA protein to the total level of p65/RelA, as measured by Western blot[36 (link)].
The activity of NFκB was measured by the ratio of the level of phosphorylation of the p65/RelA protein to the total level of p65/RelA, as measured by Western blot[36 (link)].
3
Western Blot Analysis of Cellular Proteins
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Proteins from cells were isolated using RIPA buffer (Vazyme) and the concentration was checked by Detergent Compatible Bradford Protein Quantification Kit (Vazyme). Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to segregate proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with the primary antibodies: E-cadherin (1:3000, ab40772, Abcam, Cambridge, United Kingdom), Vimentin (1:2500, ab92547, Abcam), YAP1 (1:3000, ab52771, Abcam), C3aR (1:1000, ab126250, Abcam), ICAM-1 (1:1000, ab2213, Abcam), or β-actin (1:2500, ab8227, Abcam) overnight. After being rewashed with PBS, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The membranes were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being treated with ECL kit (Vazyme).
4
Immunofluorescence Analysis of ICAM-1 Expression
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The cells cultured in 2D or onto 3D scaffolds with or without immunogenic treatment for 72 h were fixed with 4% formaldehyde in PBS. Samples grown in 2D were permeabilized in 0.1% Triton X-100 (Sigma-Aldrich, USA) and blocked in 3% BSA in PBS. Overnight incubation with primary mouse anti-ICAM1 antibody (ab2213; Abcam, UK) diluted 1:250 was followed by goat Anti-Mouse IgG H&L (Alexa Fluor 488) preadsorbed secondary antibody (ab150117; Abcam, UK) diluted 1:500 plus Alexa fluor 647 Phalloidin (A22287; Invitrogen, USA) diluted 1:40 1 h incubation. DAPI (NucBlue, USA) was added to the wells prior to analysis by confocal microscopy. Cells grown onto 3D scaffolds were stained for actin cytoskeleton (1:40 dilution; Alexa Fluor 555 phalloidin, Invitrogen, USA) and nuclear DNA (5 μM DRAQ5; eBioscience, USA). Images were captured on a confocal laser microscope (A1 Confocal Microscope, Nikon Instruments). Attached antibodies fluorescence was quantified in the confocal images using corrected total cell fluorescent intensity plug in the Image J software, and the results were statistically analyzed.
5
Renal Pathology Assessment via Immunohistochemistry
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Kidney sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and masson trichrome (MT; Richard-Allan Scientific, Kalamazoo, MI, USA) to assess the renal pathology. For immunohistochemical staining, rabbit anti-rat MCP-1 (ab25124; Abcam, Cambridge, MA, USA), IL-1β (ab82558; Abcam), IL-6 (ab6672; Abcam), mouse anti-rat TNF-α (ab1793; Abcam), ICAM-1 (ab2213; Abcam) and CD68 (ab74704; Abcam), transforming growth factor-β (TGF-β; BS1361; Bioworld Technology, Inc., St. Louis Park, MN, USA), and fibronectin (FN; ab23751; Abcam) were used as the primary antibodies and biotinylated goat anti-rabbit or mouse IgG as the secondary antibody (Dako, Glostrup, Denmark). Standard immunohistochemical procedures were then used (28 (link)). The expression levels of the above factors were quantified by Image Pro-Plus v.6.0 software via analyzing the integral optical density in 10 non-overlapping cortical fields (magnification, ×400).
6
Vascular Cell Coculture Protocol
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Primary HASMCs, primary HUVECs, SMC growth medium-2 (SmGM-2), and endothelial cell growth medium-2 (EGM-2) were supplied from Lonza (Basel, Switzerland). THP-1 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). RPMI 1640 and Dulbecco’s modified Eagle’s medium (DMEM) were ordered from Corning (New York, NY, USA), and human plasma fibronectin was purchased from Millipore (Burlington, MA, USA). Sixteen percentage formaldehyde and Hoechst 33342 were obtained from Thermo Fisher Scientific. Trichloro(1H,1H,2H,2H-perfluorooctyl)silane, erioglaucine, Triton X-100, and albumin from bovine serum (BSA) were bought from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-smooth muscle myosin heavy chain 11 antibody (ab133567, 1:25), mouse anti-CD31 antibody conjugated with Alexa Fluor 488 (ab215911, 1:100), mouse anti-intercellular adhesion molecule 1 (ICAM1) antibody (ab2213, 1:50), rabbit anti-von Willebrand factor antibody (ab6994, 1:100), goat anti-mouse IgG H&L Alexa Fluor 488 (ab150113, 1:200), and goat anti-rabbit IgG H&L Alexa Fluor 594 (ab150080, 1:200) were ordered from Abcam (Cambridge, UK). Recombinant human TNF-α was supplied from PeproTech (Rocky Hill, NJ, USA). A PDMS polymeric base and a curing agent were purchased from Dow Chemical Company (Midland, MI, USA).
7
Evaluating EMT Markers in Cell Lines
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Total protein was isolated using lysis buffer (Cell Signaling Technology) containing protease inhibitors (Pierce Biotechnology, Waltham, USA). Equal amounts of protein were loaded on a 10% SDS-PAGE gel. The polyvinylidene fluoride membranes were blotted with the following primary antibodies, including mouse anti-E-cadherin (Abcam, ab76055), mouse anti-ICAM1 (Abcam, ab2213), mouse anti-vimentin (Abcam, ab8978), rabbit anti-p-GSK3 β (Abcam, ab75745), rabbit anti-β-catenin (Abcam, ab16051), rabbit anti-wnt3a (Abcam, ab234099) and Mouse anti-GAPDH (Multi sciences, ab011-040).
After 1 h incubating with secondary antibodies, anti-Mouse IgG-HRP (Thermos, RA230188) and anti-Rabbit IgG-HRP (ab205718), the blots were visualized by the Alphalmager™ 2000 Imaging System (Alpha Innotech, San Leandro, USA). The band density was quantified using ImageJ.
After 1 h incubating with secondary antibodies, anti-Mouse IgG-HRP (Thermos, RA230188) and anti-Rabbit IgG-HRP (ab205718), the blots were visualized by the Alphalmager™ 2000 Imaging System (Alpha Innotech, San Leandro, USA). The band density was quantified using ImageJ.
8
Cytokine Profiling of Cell Supernatant
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Cells in the logarithmic growth phase were detached using 0.25% trypsin-0.02% EDTA. After centrifugation, the cells were resuspended in complete culture medium and then subjected to incubation in a 24-well plate at a density of 6 × 105 cells/well at 37° C in a 5% CO2 atmosphere. After the cells adhered to the well, the cell supernatant was collected and stored at -20° C. The levels of ICAM-1 (ab2213, Abcam, Cambridge, UK), IL-6 (ab6672, Abcam, Cambridge, UK), IL-10 (ab100549, Abcam, Cambridge, UK), and IL-1β (ab214025, Abcam, Cambridge, UK) in the cell supernatant were detected in strict accordance with the provided instructions of the ELISA kit. The experiment was repeated three times to calculate the mean values.
9
Cytotoxicity Assay of Anti-cancer Drugs
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LECs were cultured on glass coverslips in complete media as described above; the LEC monolayer was treated with 1 μM carboplatin, cisplatin, oxaliplatin, docetaxel, doxorubicin, MAZ51 (61 (link)), or appropriate solvent control (referred to as ‘vehicle’) for 6 hours (phenotypic studies) or 48 hours (live/dead analysis). After treatment, coverslips were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and underwent immunofluorescent staining with Ki67 to assess proliferation (Millipore, Cat. #AB9260); ICAM1 to assess cellular adhesion molecule expression (Abcam, Cat. #AB2213); VE-Cadherin (Abcam, Cat. #AB33168) and CD31 (R&D systems, Cat. #AF806) to assess cellular junctions. For live/dead analysis, amine-based fixable live/dead solutions (Life Technologies, Cat. #L23101) were added to cell media of living LECs after 48 hours of drug treatment and five random images were taken of each well; technical replicates were averaged to yield one biological replicate. Each quantification was performed with a minimum of 3 biological replicates.
10
Immunophenotyping of Immune Cells
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Antibodies were used following the manufacturers’ protocols. Briefly, 0.5 µg of antibody was added to 500 × 103 cells suspended in FACS buffer (90% PBS 10% FBS 0.02% Na-Azide) for 30 min on ice. Then cells were washed twice in PBS and suspended in 1.5 ml of FACS buffer (90% PBS 10% FBS 0.02% Na-Azide) containing 1 µg of secondary antibody. Cells were washed and suspended in imaging buffer (RPMI without phenol red, 10% FBS, 25 mM HEPES) for live-cell imaging.
The antibodies used in our experiments include:
Mouse anti human CD45 (BD Pharmingen, PMG555480)
Mouse monoclonal IgG1 αCD45-Alexa647 (BioLegend, 304056)
Mouse monoclonal IgG2a αCD11a (LFA1α) (BD Pharmingen, 555378)
Mouse monoclonal Anti-ICAM1 (Abcam, ab2213)
Mouse monoclonal Anti-CD80 (Abcam, ab86473)
Rabbit monoclonal Anti-CTLA4 (Abcam, ab134090)
Rabbit monoclonal Anti-CD28 (Abcam, ab243228)
Goat anti-mouse Atto488 secondary antibody (Sigma-Merck, 62197)
Goat anti-mouse IgG1 (γ1) secondary antibody, Alexa Fluor 647 conjugate (Life Technologies, A21240)
Goat anti-Rabbit secondary antibody, Alexa Fluor 647 conjugate (Life Technologies, A21244)
The antibodies used in our experiments include:
Mouse anti human CD45 (BD Pharmingen, PMG555480)
Mouse monoclonal IgG1 αCD45-Alexa647 (BioLegend, 304056)
Mouse monoclonal IgG2a αCD11a (LFA1α) (BD Pharmingen, 555378)
Mouse monoclonal Anti-ICAM1 (Abcam, ab2213)
Mouse monoclonal Anti-CD80 (Abcam, ab86473)
Rabbit monoclonal Anti-CTLA4 (Abcam, ab134090)
Rabbit monoclonal Anti-CD28 (Abcam, ab243228)
Goat anti-mouse Atto488 secondary antibody (Sigma-Merck, 62197)
Goat anti-mouse IgG1 (γ1) secondary antibody, Alexa Fluor 647 conjugate (Life Technologies, A21240)
Goat anti-Rabbit secondary antibody, Alexa Fluor 647 conjugate (Life Technologies, A21244)
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