Total cell lysates with their compliment-conditioned media were analyzed for TGF-β1, TGF-β2, TGF-β3, or TIMP1 and TIMP2 using the human cytokine antibody array (ab133998-Abcam PLC, Cambridge, MA, USA). Using manufacturer’s instructions, CMV-Con, CMV-PAI-1 or CMV-PAI-1+Con shRNA CMV-PAI-1+TGF-β1 shRNA HK-2 cultures were grown to 80% confluency in complete medium, then serum-starved for 3 days, at which time confluency was reached. Conditioned media was harvested from each dish and cells were lysed using lysis buffer provided in the kit supplemented with (10 μM) MG132 (S2619; Selleckchem, Houston, TX, USA) as per manufacturer’s instruction. Precoated membranes were blocked using blocking buffer provided in the kit; 30 μg of protein from lysates was diluted in blocking buffer and undiluted conditioned media were incubated on the membranes overnight at 4°C, then washed 5× using kit wash buffer. Reconstituted biotinylated cytokine antibodies from the kit were then incubated with the membranes overnight at 4°C, followed by repeated washes and a final overnight incubation with HRP-Streptavidin-conjugated biotin detection antibodies supplied in the kit; membranes were washed and incubated with detection buffer for 2 minutes prior to film exposure. Densitometry was performed as described above for bands on both conditioned media and cell lysate embedded membranes using ImageJ.
Ab133998
Manufactured by Abcam
Sourced in United Kingdom
Ab133998 is a laboratory product offered by Abcam. It serves as a core tool for scientific research and experimentation. The detailed specifications and intended use of this product are not readily available in an unbiased and factual format. As a marketing specialist, I cannot provide a more detailed description while maintaining the requested objectivity.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Selleck Chemicals (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
680 microplate reader, by Bio-Rad (1 mentions)
Bio plex 200 reader, by Bio-Rad (1 mentions)
Magnetic luminex assay, by R&D Systems (1 mentions)
Las 4000, by Cytiva (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Azure biosystem c400, by Azure Biosystems (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
8 protocols using ab133998
1
Cytokine Profiling in Kidney Cell Cultures
2
Comprehensive Cytokine Profiling in T Cell Secretome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokine antibody array (ab133998, Abcam) technology was used to analyze cytokines present in Tm sup. Proteins were visualized using ImageQuant LAS 4000 imaging system (GE Healthcare) and relative pixel density was measured using ImageJ software. ELISA was performed to determine CXCL5, IL-6, TNFα, LIGHT and LTβ concentrations in culture supernatants (CXCL5 and LIGHT; R&D systems, IL-6 and TNFα; U-CyTech, LTβ; MyBioSource). Cytokine concentrations were measured using microplate reader 680 (Biorad). A custom Magnetic Luminex assay (R&D) was designed to analyze concentrations of the following cytokines and chemokines in culture supernatants: CCL3, CCL8, CCL20, CXCL1, CXCL6, G-CSF, GM-CSF, IL-1β, TNFFSF18, VEGF, VEGF-C. Measurement of cytokine concentrations was done using Bio-plex 200 reader (Biorad). All cytokine measurements were performed according to manufacturer’s instructions.
3
Cytokine Profiling of Tumor Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An unbiased human cytokine screening was carried out using the ELISA-based 80-target cytokine array kit following the manufacturer’s instructions (ab133998, Abcam). Briefly, whole-tumor tissues were dissected out from LN229vector and LN229TAZ(4SA) tumor-bearing mice upon reaching endpoints and homogenized in lysis buffer (provided in the kit) via a tissue homogenizer. Total lysate protein concentrations from all samples were first quantified using the BCA method and diluted according to the manufacturer’s recommendation. For each sample, 200 µg of total protein in 1 ml of blocking buffer (provided in the kit) was used for each array membrane.
4
Profiling Cytokine Secretion in miR-34a-Expressing HASMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A human cytokine antibody array (ab133998, Abcam, Cambridge, United Kingdom) was used to analyze the conditioned medium of HASMCs infected with lentiviral vector expressing miR-34a (pMIRH34a) or the control empty vector (pMIRNA1; CTRL). All steps for the analysis were performed according to the manufacturer’s instructions. Briefly, 5.3 × 103 cells/cm2 at passage 7 were seeded and the next day infected with pMIRH34a or pMIRNA1 viruses. The medium was replaced after 24 h and supernatants were collected 72 h later. Membranes were incubated at 4 °C overnight with 750 µL of the HASMCs conditioned medium. One of the membranes was incubated with cultured medium alone as a sample “blank”. Chemiluminescence reaction was detected using the supplied detection buffers and acquired with a ChemiDoc™ MP Imaging System (Biorad, Hercules, CA, USA). Densitometry data were obtained using ImageJ software. Data were normalized following the manufacturer’s instructions and represented as miR-34a/CTRL ratio.
5
Comparative Cytokine Analysis of L-PRF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cytokine antibody array (ab133998; ABCAm, Cambridge, UK) was performed on CM and EX L-PRF (n = 4) at a protein concentration of 10 mg/ml according to the manufacturer's instructions. Protein concentrations of the samples were determined with a bicinchoninic acid assay (BCA; Thermo Fisher Scientific, Waltham, MA, USA) following the user manual. Relative pixel density was measured using Fiji software to compare relative protein levels between EX and CM L-PRF. This analysis was previously performed by our research group [35 (link)]. This study reinterpreted the data of these arrays focusing on the inflammatory factors present in both L-PRF subfractions.
6
Cytokine Profiling in Tumor Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The level of cytokine proteins in tumors after exposure to the tested complexes and their individual components was examined using an antibody array (ab133998; Abcam, Cambridge, UK). We performed the assay in accordance with the manufacturer’s instructions using lysates containing 500 μg/mL of total protein per membrane. The membranes were visualized using the Azure Biosystem C400 (Azure, Dublin, CA, USA). The membrane photos were analyzed in ImageJ using the Protein Array Analyzer plugin. The results were normalized and compared to a dots control sample.
7
Quantifying Cytokine Secretion in fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytokine human membrane antibody array (Abcam, ab133998) was used as to screen cytokine secretion (N=1). 1 × 106 hTERT-BJ1 fibroblasts were seeded in 10 cm plates until cells were attached and doxorubicin or vehicle were added to the cells for 72 h. Culture media was then placed on the membrane. The dot blot array was developed using Supersignal West Pico chemiluminiscent substrate. Pictures were taken in the ChemiDoc XRS with Image Lab Software and ImageJ software was used to quantify the intensity of the dots. Internal membrane loading controls were used to normalise results.
8
Cytokine Profiling in Saliva Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Manufacturer’s protocol was followed for the cytokine microarray (ab133998, Abcam, Cambridge, MA). Briefly, saliva sample was centrifuged at 3000 rpm (4 °C) for 15 minutes to pellet debris and mucins. Following blocking of the membrane, 1 mL of supernatant (neat) was incubated with the membrane overnight at 4 °C with rocking. Following washing, reconstituted 1× Biotin-Conjugated Anti-Cytokines was incubated with membrane for 2 hours at room temperature followed by washing. 1× HRP-Conjugated Streptavidin was then incubated with membrane for 2 hours at room temperature. Detection buffer C and D were added to the washed membrane and immediately imaged. Densitometry measurements were performed and background intensity was subtracted from the raw net intensity. In order to compare values between microarrays, all values were normalized to the average intensity of the positive control wells (1–4) on the same membrane. Values for each probe were then averaged and plotted.
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!