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Cell Extracts

Cell Extracts are preparations obtained by the disruption or lysis of cells, which contain the intracellular contents including organelles, proteins, nucleic acids, and other biomolecules.
These extracts are widely used in biochemical and molecular biology research to study cellular processes, protein interactions, and enzyme activities.
The composition and properties of cell extracts can be optimized through careful selection of extraction methods and conditions to enhance the accuracy and reproducibility of experiments.
PubCompare.ai's AI-driven platform empowers researchers to identify the most effective cell extract protocols from literature, preprints, and patents, leveraging cutting-edge comparisons to pinpoint the most accurate and reproducible techniques.
Ths platform helps take your cell extract research to new heights by enhancing experimental accuracy and reproducibilty.

Most cited protocols related to «Cell Extracts»

1
Integrative Epigenomic Analysis of 127 Consolidated Genomes
All genome-wide maps of histone modifications, DNA accessibility, DNA methylation and RNA expression are freely available online. Raw sequencing data deposited at the Short Read Archive or dbGAP is linked from http://www.ncbi.nlm.nih.gov/geo/roadmap/epigenomics/. All primary processed data (including mapped reads) for profiling experiments are contained within Release 9 of the Human Epigenome Atlas (http://genboree.org/EdaccData/Release-9/). Complete metadata associated with each dataset in this collection is archived at GEO and describes samples, assays, data processing details and quality metrics collected for each profiling experiment.
Release 9 of the compendium contains uniformly pre-processed and mapped data from multiple profiling experiments (technical and biological replicates from multiple individuals and/or datasets from multiple centers). In order to reduce redundancy, improve data quality and achieve uniformity required for our integrative analyses, experiments were subjected to additional processing to obtain comprehensive data for 111 consolidated epigenomes (See methods sections below for additional details). Numeric epigenome identifiers EIDs (e.g. E001) and mnemonics for epigenome names were assigned for each of the consolidated epigenomes. Table S1 (QCSummary sheet) summarizes the mapping of the individual Release 9 samples to the consolidated epigenome IDs. Key metadata such as age, sex, anatomy, epigenome class (see below), ethnicity and solid/liquid status were summarized for the consolidated epigenomes. Datasets corresponding to 16 cell-lines from the ENCODE project (with epigenome IDs ranging from E114-E129) were also used in the integrative analyses23 (link). All datasets from the 127 consolidated epigenomes were subjected to processing filters to ensure uniformity in terms of read length based mappability and sequencing depth as described below.
Each of the 127 epigenomes included consolidated ChIP-seq datasets for a core set of histone modifications - H3K4me1, H3K4me3, H3K27me3, H3K36me3, H3K9me3 as well as a corresponding whole-cell extract sequenced control. 98 epigenomes and 62 epigenomes had consolidated H3K27ac and H3K9ac histone ChIP-seq datasets respectively. A smaller subset of epigenomes had ChIP-seq datasets for additional histone marks, giving a total of 1319 consolidated datasets (Table S1, QCSummary sheet). 53 epigenomes had DNA accessibility (DNase-seq) datasets. 56 epigenomes had mRNA-seq gene expression data. For the 127 consolidated epigenomes, a total of 104 DNA methylation datasets across 95 epigenomes involved either bisulfite treatment (WGBS or RRBS assays) or a combination of MeDIP-seq and MRE-seq assays. In addition to the 1936 datasets analyzed here across 111 reference epigenomes, the NIH Roadmap Epigenomics Project has generated an additional 869 genome-wide datasets, linked from GEO, the Human Epigenome Atlas, and NCBI, and also publicly and freely available.
Kundaje A., Meuleman W., Ernst J., Bilenky M., Yen A., Kheradpour P., Zhang Z., Heravi-Moussavi A., Liu Y., Amin V., Ziller M.J., Whitaker J.W., Schultz M.D., Sandstrom R.S., Eaton M.L., Wu Y.C., Wang J., Ward L.D., Sarkar A., Quon G., Pfenning A., Wang X., Claussnitzer M., Coarfa C., Harris R.A., Shoresh N., Epstein C.B., Gjoneska E., Leung D., Xie W., Hawkins R.D., Lister R., Hong C., Gascard P., Mungall A.J., Moore R., Chuah E., Tam A., Canfield T.K., Hansen R.S., Kaul R., Sabo P.J., Bansal M.S., Carles A., Dixon J.R., Farh K.H., Feizi S., Karlic R., Kim A.R., Kulkarni A., Li D., Lowdon R., Mercer T.R., Neph S.J., Onuchic V., Polak P., Rajagopal N., Ray P., Sallari R.C., Siebenthall K.T., Sinnott-Armstrong N., Stevens M., Thurman R.E., Wu J., Zhang B., Zhou X., Beaudet A.E., Boyer L.A., De Jager P., Farnham P.J., Fisher S.J., Haussler D., Jones S., Li W., Marra M., McManus M.T., Sunyaev S., Thomson J.A., Tlsty T.D., Tsai L.H., Wang W., Waterland R.A., Zhang M., Chadwick L.H., Bernstein B.E., Costello J.F., Ecker J.R., Hirst M., Meissner A., Milosavljevic A., Ren B., Stamatoyannopoulos J.A., Wang T, & Kellis M. (2015). Integrative analysis of 111 reference human epigenomes. Nature, 518(7539), 317-330.
Publication 2015
Biological Assay Biopharmaceuticals Cell Extracts Cell Lines Chromatin Immunoprecipitation Sequencing Deoxyribonuclease I DNA Methylation Epigenome Ethnicity Gene Expression Genome Histone Code histone H3 trimethyl Lys4 Histones Homo sapiens hydrogen sulfite RNA, Messenger Transcription, Genetic
2
Ribosome and mRNA Profiling of 4EBP1/2 DKO MEFs
To generate ribosome and mRNA profiling libraries, WT MEFs (4EBP1/2+/+; p53−/−) or DKO MEFS (4EBP1/2−/−; p53−/−) were treated with vehicle or 250 nM Torin1 for 2 h. Cellular extracts were partitioned for either ribosome profiling or mRNA profiling. Small RNA libraries were prepared according to established protocols8 with some modifications, and analyzed by high-throughput sequencing. Transcript abundance was determined through an iterative alignment and mapping strategy to a non-redundant library of mouse transcripts based on Refseq definitions.
Thoreen C.C., Chantranupong L., Keys H.R., Wang T., Gray N.S, & Sabatini D.M. (2012). A unifying model for mTORC1-mediated regulation of mRNA translation. Nature, 485(7396), 109-113.
Publication 2012
Cell Extracts DNA Library EIF4EBP1 protein, human Mus Ribosomes RNA, Messenger
3
Metabolomics Analysis of Viral Infection
Full scan LC/MS (m/z range 85–2000) was performed essentially as previously described [8] . Cell extracts or supernatants were treated with acetonitrile (2∶1, v/v) and centrifuged at 14,000× g for 5 min at 4°C to remove proteins. Samples were maintained at 4°C in an autosampler until injection. A Thermo Orbitrap-Velos mass spectrometer (Thermo Fisher, San Diego, CA) coupled with anion exchange chromatography was used for data collection, via positive-ion electrospray ionization (ESI). Metabolites of interest were identified by tandem mass spectrometry on a LTQ-FTMS, where the biological sample, biological sample spiked in with authentic chemical and authentic chemical reference were run sequentially. The and were done in the ion trap of the LTQ-FTMS, with an isolation width of 1 amu and a normalized collision energy of 35 eV.
The LC/MS data were processed with apLCMS program [25] (link) for feature extraction and quantification. Significant features were also verified by inspecting the raw data (Figure S5). Features were removed if their intensity is below 10,000 in every sample class. Missing intensity values were imputed to 1000. The intensities were log2 transformed. Low quality features were further filtered out if their averaged in-class coefficient of variation was greater than 0.2. Averaged ion intensity over three machine replicates was used for subsequent analysis. These 7,995 features constituted the reference list . No normalization was used because total ion counts in all samples were very similar. Student's t-test was used to compare infected samples (at 6 hr) versus mock infections (at 6 hr), and infected samples (at 6 hr) versus baseline controls (at 0 hr). Features with in both tests were included in the significant list . The feature table, , and predictions are given in Dataset S1.
Li S., Park Y., Duraisingham S., Strobel F.H., Khan N., Soltow Q.A., Jones D.P, & Pulendran B. (2013). Predicting Network Activity from High Throughput Metabolomics. PLoS Computational Biology, 9(7), e1003123.
Publication 2013
acetonitrile Anions Biopharmaceuticals Cell Extracts Chromatography Infection isolation Proteins Radionuclide Imaging Tandem Mass Spectrometry
4
Comparative Evaluation of Amino Acid Profiling Methods
Technical consistency (i.e. reproducibility and linearity) of the 3AA method was compared to two conventional chromatographic separations used for small molecule and amino acid analyses. Pancreatic cancer cell extracts (biological triplicates) either undiluted or diluted five-fold, were analyzed using (i) the 3AA method, as well as (ii) a 15 min gradient on a Kinetex HILIC column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) and (iii) a 23 min gradient on an Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA). In (ii), samples were analyzed on the Kinetex HILIC at 350 μl/min (mobile phases: A: ACN; B: 18 mΩ H2O, 20 mM (NH4)2CO3, 0.1% NH4OH; gradient: 1.5 min hold at 5% B; 5-60% B in 8.5 min; 60-95% B in 0.5 min at 0.5 ml/min; 95% hold for 2 min; 95-5% B in 0.5 min; 5 hold for 2 min; column temperature: 25°C). In (iii), samples were analyzed on the Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA) at 500 μl/min (mobile phases: A: 18 mΩ H2O, 20 mM (NH4)2CO3 pH 4.00; B: acetonitrile; gradient: 3 min hold 85% B; 85-30% B in 9 min; 30-2% B in 3 min; 2-85% of B in 1 min; 85% equilibration for 7 min; column temperature: 60°C).
The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs.
MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012 ). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Relative quantitation was performed by exporting integrated peak areas values into Excel (Microsoft, Redmond, CA, USA) for statistical analysis including T-Test and ANOVA (significance threshold for p-values < 0.05) and unsupervised Principal Component Analysis (PCA) (Pan et al. 2007 (link); Fonville et al. 2010 ), calculated through the MultiBase macro (freely available at www.NumericalDynamics.com).
Nemkov T., D'Alessandro A, & Hansen K.C. (2015). Three Minute Method for Amino Acid Analysis by UHPLC and high resolution quadrupole orbitrap mass spectrometry. Amino acids, 47(11), 2345-2357.
Publication 2015
acetonitrile Amides Amino Acids Biopharmaceuticals Cell Extracts Chemical Processes Chromatography Isotopes neuro-oncological ventral antigen 2, human Pancreatic Carcinoma Retention (Psychology)
5
Proteomic analysis of ubiquitinated proteins

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Publication 2011
Buffers Cell Extracts Cells HEK293 Cells Light Peptides Sepharose Staphylococcal Protein A Tandem Mass Spectrometry Trypsin

Most recents protocols related to «Cell Extracts»

1
Cytotoxicity of Plant Stem Cell Extracts
Not available on PMC !

Example 7

The MTT Cell Proliferation assay determines cell survival following apple stem cell extract treatment. The purpose was to evaluate the potential anti-tumor activity of apple stem cell extracts as well as to evaluate the dose-dependent cell cytotoxicity.

Principle: Treated cells are exposed to 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTT enters living cells and passes into the mitochondria where it is reduced by mitochondrial succinate dehydrogenase to an insoluble, colored (dark purple) formazan product. The cells are then solubilized with DMSO and the released, solubilized formazan is measured spectrophotometrically. The MTT assay measures cell viability based on the generation of reducing equivalents. Reduction of MTT only occurs in metabolically active cells, so the level of activity is a measure of the viability of the cells. The percentage cell viability is calculated against untreated cells.

Method: A549 and NCI-H520 lung cancer cell lines and L132 lung epithelial cell line were used to determine the plant stem cell treatment tumor-specific cytotoxicity. The cell lines were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a 5% CO2 at 37 Celsius. Cells were seeded at 5×103 cells/well in 96-well plates and incubated for 48 hours. Triplicates of eight concentrations of the apple stem cell extract were added to the media and cells were incubated for 24 hours. This was followed by removal of media and subsequent washing with the phosphate saline solution. Cell proliferation was measured using the MTT Cell Proliferation Kit I (Boehringer Mannheim, Indianapolis, IN) New medium containing 50 μl of MTT solution (5 mg/ml) was added to each well and cultures were incubated a further 4 hours. Following this incubation, DMSO was added and the cell viability was determined by the absorbance at 570 nm by a microplate reader.

In order to determine the effectiveness of apple stem cell extracts as an anti-tumor biological agent, an MTT assay was carried out and IC50 values were calculated. IC50 is the half maximal inhibitory function concentration of a drug or compound required to inhibit a biological process. The measured process is cell death.

Results: ASC-Treated Human Lung Adenocarcinoma Cell Line A549.

TABLE 7
Results of cytotoxicity of apple stem cell extract on lung cancer cell
line A549 as measured by MTT assay (performed in triplicate).
Values of replicates are % of cell death.
Concentration*replicatereplicatereplicateMean of% Live
(μg/ml)123replicatesSDSEMCells
25093.1890.8690.3491.461.510.878.54
10086.8885.1885.6985.920.870.5014.08
5080.5879.4981.0480.370.800.4619.63
2574.2873.8176.3974.831.380.7925.17
12.567.9868.1371.7569.282.131.2330.72
6.2561.6762.4567.1063.742.931.6936.26
3.12555.3756.7762.4558.203.752.1641.80
1.56249.0751.0857.8052.654.572.6447.35
0.78142.7745.4053.1547.115.403.1252.89

Results: ASC-Treated Human Squamous Carcinoma Cell Line NCI-H520.

TABLE 8
Results of cytotoxicity of apple stem cell extract on lung cancer
cell line NCI-H520 measured by MTT assay (performed in triplicate).
Values of replicates are % of cell death.
Concen-%
tration*replicatereplicatereplicateMean ofLive
(μg/ml)123replicatesSDSEMcell
25088.2889.2987.7388.430.790.4611.57
10078.1379.1978.1378.480.610.3521.52
5067.9869.0968.5468.540.560.3231.46
2557.8358.9958.9458.590.660.3841.41
12.547.6848.8949.3448.640.860.5051.36
6.2537.5338.7939.7538.691.110.6461.31
3.12527.3728.6930.1528.741.390.8071.26
1.56217.2218.5920.5618.791.680.9781.21
0.781 7.07 8.4810.96 8.841.971.1491.16

Results: ASC-treated Lung Epithelial Cell Line L132.

TABLE 9
Results of cytotoxicity of apple stem cell extract on
lung epithelial cell line L132 as measured by MTT assay
(performed in triplicate). Values of replicates are % of cell death.
Concen-rep-rep-rep-Mean%
tration*licatelicatelicateofLive
(μg/ml)123replicatesSDSEMcell
25039.5142.5244.0342.022.301.3357.98
10032.9334.4433.6933.690.750.4466.31
5030.6028.9430.5230.020.940.5469.98
2527.9627.8127.1327.630.440.2572.37
12.525.6225.5525.4025.520.120.0774.48
6.2523.1320.8718.6120.872.261.3179.13
3.12513.3411.0811.8312.081.150.6687.92
1.562 6.56 7.31 9.57 7.811.570.9192.19
0.781 8.06 4.30 3.54 5.302.421.4094.70

Summary Results: Cytotoxicity of Apple Stem Cell Extracts.

TABLE 10
IC50 values of the apple stem cell extracts on the on the target
cell lines as determined by MTT assay.
Target Cell
LineIC50
A54912.58
NCI-H52010.21
L132127.46

Apple stem cell extracts killed lung cancer cells lines A549 and NCI-H520 at relatively low doses: IC50s were 12.58 and 10.21 μg/ml respectively as compared to 127.46 μg/ml for the lung epithelial cell line L132. Near complete anti-tumor activity was seen at a dose of 250 μg/ml in both the lung cancer cell lines. This same dose spared more than one half of the L132 cells. See Tables 7-10. The data revealed that apple stem cell extract is cytotoxic to lung cancer cells while sparing lung epithelial cells. FIG. 6 shows a graphical representation of cytotoxicity activity of apple stem cell extracts on lung tumor cell lines A549, NCIH520 and on L132 lung epithelial cell line (marked “Normal”). The γ-axis is the mean % of cells killed by the indicated treatment compared to unexposed cells. The difference in cytotoxicity levels was statistically significant at p≤05.

Example 9

The experiment of Example 7 was repeated substituting other plant materials for ASC. Plant stem cell materials included Dandelion Root Extract (DRE), Aloe Vera Juice (AVJ), Apple Fiber Powder (AFP), Ginkgo Leaf Extract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells (OSC) as described in Examples 1 and 2. The concentrations of plant materials used were nominally 250, 100, 50, 25, 6.25, 3.125, 1.562, and 0.781 μg/mL. These materials were tested only for cells the human lung epithelial cell line L132 (as a proxy for normal epithelial cells) and for cells of the human lung adenocarcinoma cell line A549 (as a proxy for lung cancer cells).

A549 cells lung cancer cell line cytotoxicity results for each of the treatment materials.

DRE-Treated Lung Cancer Cell Line A549 Cells.

TABLE 11
Triplicate results of cell death of DRE-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration%
(μg/mL)-DRE-Live
treated A549% of cell deathMeanSDSEMcell
25080.4376.4074.8477.232.891.6722.77
10067.6075.2663.7768.885.853.3831.12
5065.3262.9459.9462.732.701.5637.27
2556.8357.9748.1454.315.383.1145.69
6.2555.5949.6949.1751.483.572.0648.52
3.12551.7648.4545.3448.523.211.8551.48
1.56243.6944.0036.0241.244.522.6158.76
0.78137.4726.1919.5727.749.055.2372.26

AVJ-Treated Lung Cancer Cell line A549 Cells.

TABLE 12
Triplicate results of cell death of AVJ-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration%
(μg/mL)-AVJ-treatedLive
A549% of cell deathMeanSDSEMcell
25076.8178.1675.8876.951.140.6623.05
10076.4075.2673.7175.121.350.7824.88
5065.3266.1559.9463.803.371.9536.20
2550.1048.4556.6351.734.322.5048.27
6.2547.5246.3846.1746.690.720.4253.31
3.12539.8638.6143.7940.752.701.5659.25
1.56232.4019.7730.5427.576.823.9472.43
0.78120.5015.6332.1922.778.514.9277.23

AFP-Treated Lung Cancer Cell line A549 Cells.

TABLE 13
Triplicate results of cell death of AFP-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration%
(μg/mL)-AFP-treatedLive
A549% of cell deathMeanSDSEMcell
25086.1387.9986.6586.920.960.5613.08
10079.5081.0682.0980.881.300.7519.12
5073.6072.4671.3372.461.140.6627.54
2568.0167.7066.9867.560.530.3132.44
6.2560.8762.1160.7761.250.750.4338.75
3.12549.4851.7650.7250.661.140.6649.34
1.56240.0641.7247.0042.933.622.0957.07
0.78139.2337.7836.8537.961.200.6962.04

GLE-treated Lung Cancer Cell line A549 Cells.

TABLE 14
Triplicate results of cell death of GLE-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration%
(μg/mL)-GLE-treatedLive
A549% of cell deathMeanSDSEMcell
25088.4291.4990.4490.121.560.909.88
10084.3983.7783.1683.770.610.3516.23
5079.4781.5876.7579.272.421.4020.73
2573.6072.5471.4072.511.100.6327.49
6.2562.8963.6859.9162.161.991.1537.84
3.12550.1854.4751.8452.162.171.2547.84
1.56246.9344.3043.3344.851.861.0755.15
0.78139.5639.3940.9639.970.870.5060.03

LSC-treated lung cancer cell lines A549 cells.

TABLE 15
Triplicate results of cell death of LSC-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration
(μg/mL)% Live
LSC treated A549% of cell deathMeanSDSEMcell
25077.5478.8578.2078.200.650.3821.80
10077.1476.0476.5976.590.550.3223.41
5066.4268.5266.8267.251.120.6532.75
2559.8067.2264.1663.733.732.1536.27
6.2550.5348.8248.0749.141.260.7350.86
3.12541.1443.6042.7242.491.240.7257.51
1.56239.4739.7440.6139.940.600.3460.06
0.78138.5531.8336.7935.723.482.0164.28

OSC-treated Lung Cancer Cell line A549 Cells.

TABLE 16
Triplicate results of cell death of OSC-treated
A549 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration
(μg/mL)% Live
OSC-treated A549% of cell deathMeanSDSEMcell
25070.8465.5771.4969.303.251.8730.70
10048.8150.9157.2852.334.412.5547.67
5046.5949.6053.3349.843.381.9550.16
2538.7740.8136.5838.722.111.2261.28
6.2535.7440.7941.0539.193.001.7360.81
3.12534.5533.6837.0235.081.731.0064.92
1.56233.8633.4427.6331.643.482.0168.36
0.78121.3220.0034.8225.388.214.7474.62

L132 cells (“normal” lung epithelial cell line) cytotoxicity results for each of the treatment materials.

DRE-Treated Lung Epithelial Cell Line L132 cells.

TABLE 17
Triplicate results of cell death of DRE-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates.
Concentration% of %
(μg/mL)cellLive
DRE-treated L132deathMeanSDSEMcell
25086.6686.6186.6686.640.030.0213.36
10076.2977.3976.8476.840.550.3223.16
5065.9268.1767.0167.031.130.6532.97
2555.5458.9557.1957.231.700.9842.77
6.2545.1749.7347.3747.422.281.3252.58
3.12534.8040.5037.5437.612.851.6562.39
1.56224.4231.2827.7227.813.431.9872.19
0.78114.0522.0617.8918.004.012.3182.00

AVJ-Treated Lung Epithelial Cell Line L132 cells.

TABLE 18
Triplicate results of cell death of AVJ-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates
AFP-treated lung epithelial cell line L132 cells.
Concentration % of %
(μg/mL)cellLive
AVJ-treated L132deathMeanSDSEMcell
25057.0355.9353.6255.531.741.0044.47
10050.9949.7847.0449.272.031.1750.73
5044.9543.6340.4543.012.311.3456.99
2538.9137.4933.8636.752.601.5063.25
6.2532.8831.3427.2830.502.891.6769.50
3.12526.8425.1920.6924.243.181.8475.76
1.56220.8019.0514.1117.983.472.0082.02
0.78114.7612.90 7.5211.733.762.1788.27

AFP-Treated Lung Epithelial Cell Line L132 cells.

TABLE 19
Triplicate results of cell death of AFP-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates
AFP-treated lung epithelial cell line L132 cells.
Concentration
(μg/mL)% Live
AFP-treated L132% of cell deathMeanSDSEMcell
25056.1555.4357.1956.260.880.5143.74
10049.9548.2447.6448.611.200.6951.39
5043.7441.0538.0940.962.831.6359.04
2537.5433.8628.5433.324.532.6166.68
6.2531.3426.6718.9925.676.243.6074.33
3.12525.1419.489.4418.027.954.5981.98
1.56218.9412.2910.8714.034.312.4985.97
0.78112.73 5.10 6.81 8.214.002.3191.79

GLE-Treated Lung Epithelial Cell Line L132 cells.

TABLE 20
Triplicate results of cell death of GLE-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates
AFP-treated lung epithelial cell line L132 cells.
Concentration
(μg/mL)% Live
GLE-treated L132% of cell deathMeanSDSEMcell
25084.4283.2083.0883.570.740.4316.43
10080.0579.2978.5979.310.730.4220.69
5072.7571.5974.1072.811.260.7227.19
2580.0581.8679.9980.631.060.6119.37
6.2568.2670.1368.2668.881.080.6231.12
3.12560.6263.0760.6261.441.410.8238.56
1.56248.0748.7748.8348.560.420.2451.44
0.78146.2745.5746.6746.170.560.3253.83

LSC-Treated Lung Epithelial Cell Line L132 cells.

TABLE 21
Triplicate results of cell death of LSC-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates
AFP-treated lung epithelial cell line L132 cells.
Concentration
(μg/mL)% Live
LSC-treated L132% of cell deathMeanSDSEMcell
25086.4185.8285.7686.000.350.2014.00
10081.2181.2779.9980.820.720.4219.18
5075.9674.7473.5174.741.230.7125.26
2574.7472.7571.4772.991.650.9527.01
6.2570.1368.3268.2668.901.060.6131.10
3.12554.0358.0553.4455.172.511.4544.83
1.56253.9751.9851.9852.641.150.6647.36
0.78146.7945.6244.9245.78 0.940.54 54.22

OSC-Treated Lung Epithelial Cell Line L132 cells.

TABLE 22
Triplicate results of cell death of OSC-treated
L132 cells measured by MTT assay.
Percentage of live cells calculated as 100% − Mean of triplicates
AFP-treated lung epithelial cell line L132 cells.
Concentration %
(μg/mL)Live
OSC-treated L132% of cell deathMeanSDSEMcell
25061.8462.3760.4461.551.000.5738.45
10054.1453.4452.1053.231.040.6046.77
5042.9442.3040.3241.851.370.7958.15
2535.9434.4833.3134.581.320.7665.42
6.2533.9632.6732.0332.890.980.5767.11
3.12527.4826.2026.7226.800.650.3773.20
1.562 9.80 7.29 7.35 8.151.430.8391.85
0.781 7.29 8.98 8.05 8.110.850.4991.89

Calculated values.

TABLE 23
Calculated IC50 doses (ug/mL) and therapeutic ratios
(IC50 for L132 cells/IC50 for A549 cells) for each
treatment material. Values greater than one indicate
that a material would be more selective in killing cancer
cells than normal cells. ASC results imported from
Example 8. These studies indicate that at least
some of the materials may be effective anti-cancer agents.
ASC has outstanding selectivity compared to other materials.
ASCDREAVJAFPGLELSCOSC
A549 12.589.82211.4811.9811.1 13.733.9 
IC50
L132 127.4656.88 62.6682.6577.6369.26715.38
IC50
Ther.10.15.8 5.56.97.0 0.70.5
Ratio

US11872258B2. Oral cavity treatments (2024-01-16). None [US]. Inventors: Maryam Rahimi [US].
Patent 2024
14-3-3 Proteins 43-63 61-26 A549 Cells Action Potentials Adenocarcinoma of Lung Aloe Aloe vera Antineoplastic Agents Biological Assay Biological Factors Biological Processes Bromides Cardiac Arrest Cell Death Cell Extracts Cell Lines Cell Proliferation Cells Cell Survival Cytotoxin diphenyl DNA Replication Epistropheus Epithelial Cells Fibrosis Formazans Genetic Selection Ginkgo biloba Ginkgo biloba extract Homo sapiens Lingonberry Lung Lung Cancer Lung Neoplasms Malignant Neoplasms Mitochondria Mitochondrial Inheritance Neoplasms Neoplastic Stem Cells Oral Cavity PEG SD-01 Penicillins Pharmaceutical Preparations Phosphates Plant Cells Plant Leaves Plant Roots Plants Powder Psychological Inhibition Saline Solution SD 31 SD 62 SEM-76 Squamous Cell Carcinoma Stem, Plant Stem Cells Streptomycin Succinate Dehydrogenase Sulfoxide, Dimethyl Taraxacum Tetrazolium Salts
2
Anti-inflammatory Effects of Apple Stem Cell Extracts
Not available on PMC !

Example 6

In an inflammatory reaction, activated cells (such as macrophages) release a variety of pro-inflammatory cytokines (such as tumor necrosis factor alpha (TNF-α). The released cytokines can be assayed as a measure of inflammatory activity. To evaluate the anti-inflammatory role of apple stem cell extracts, mouse RAW 264.7 cell lines mouse macrophages were used as an adherent monolayer on petri dishes. These cells could be harvested easily without damage caused by enzymes or cell scrapers. The macrophages were stimulated in suspension with lipopolysaccharide (LPS) to initiate an inflammatory response. Cells were seeded into 12-well cell culture plates containing the apple stem cell extract treatment materials. After 16-18 hours, the medium conditioned by the macrophages was harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA) by measuring TNF-α levels.

Method: Three concentration of ASC (6.25, 12.5 and 25 μg/mL in media) were tested for the anti-inflammatory effect. RAW 264.7 mouse macrophage cells were maintained in DMEM containing Glutamax supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). The macrophages treated with LPS (1:500 dilution of a 0.1 mg/ml solution of LPS in phosphate buffered saline (PBS)) to produce a pro-inflammatory response. The ASC treatment was performed with a final concentration of 1×105 macrophages in wells of a 12-well plate. The cytokine assay was performed using a TNF-α ELISA from R&D Systems of Minneapolis, Minnesota.

Results indicated (Table 6, FIG. 5) that LPS alone produced an inflammatory response more than 1000 times that of unstimulated cells as measured by TNF-α expression. Treatment with ASC on the induced macrophages showed a dose-dependent decrease of TNF-α expression. ASC concentrations of 6.25, 12.5, and 25 μg/mL reduced TNF-α activity in the induced cells by 72.1, 92.1 and 94.5%, respectively. This reduced TNF-α at doses of 12.5 and 25 μg/ml was statistically significant with p≤0.05 for 25 μg/ml and p≤0.02 in 12.5 μg/ml. The apple stem cell extracts thus exerted an anti-inflammatory effect on the activated macrophage cells.

TABLE 6
Results of TNF-α release assay showing anti-inflammatory effects of apple
stem cell extracts on mouse RAW 264.7 macrophage cell line cells.
Values shown are averages of three sets of experiments.
ASC extracts dramatically reduced inflammatory responses in the target
cells, as exemplified by reduced TNF-α release
(greater inhibition of inflammation).
Apple Stem percent
Cell Extract TNF-α inhibition
Conc. (μg/ml)(pg/ml)vs. LPS
25481.8994.5
12.5687.992.1
6.252432.8972.1
LPS8712.630
unstimulated6.45

US11872258B2. Oral cavity treatments (2024-01-16). None [US]. Inventors: Maryam Rahimi [US].
Patent 2024
Anti-Inflammatory Agents Biological Assay Cell Culture Techniques Cell Extracts Cells Cytokine Enzyme-Linked Immunosorbent Assay Enzymes Hyperostosis, Diffuse Idiopathic Skeletal Inflammation Lipopolysaccharides Macrophage Mus Penicillins Phosphates Psychological Inhibition RAW 264.7 Cells Saline Solution Stem, Plant Stem Cells Streptomycin Technique, Dilution Tumor Necrosis Factor-alpha
3
Protein Extraction and Western Blot Analysis
Cell extracts were prepared in lysis buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5% sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/µL leupeptin, protease inhibitors (#P8340; Sigma-Aldrich) and phosphatase inhibitors (#P5726; Sigma-Aldrich). Protein concentrations were determined using the BCA reagent as described earlier, and samples were denatured using SDS sample buffer (#1610747; BioRad). Samples were loaded into a Criterion Tris-Glycine Extended Gel (#5671124; BioRad) and separated by electrophoreses at 60 mA. The gels were then transferred onto a nitrocellulose membrane (#1620115; BioRad) by a wet transfer system (BioRad) at 100V for 1 h at room temperature. All membranes were then blocked by incubation with 5% dry milk in TBST (TBS with 0.1% Tween20) for 1 h at room temperature. Membranes were probed with the primary antibody overnight at 4°C in the blocking buffer, washed with TBST, and incubated with the peroxidase-conjugated secondary antibody. Enhanced chemiluminescence (ECL) Western blotting substrates (#170-5061; BioRad) were used for the visualization of the results. The acquisition of images was performed using the ChemiDoc MP Imaging System (BioRad).
Hurwitz E., Parajuli P., Ozkan S., Prunier C., Nguyen T.L., Campbell D., Friend C., Bryan A.A., Lu T.X., Smith S.C., Razzaque M.S., Xu K, & Atfi A. (2023). Antagonism between Prdm16 and Smad4 specifies the trajectory and progression of pancreatic cancer. The Journal of Cell Biology, 222(4), e202203036.
Publication 2023
beta-glycerol phosphate Buffers Cell Extracts Chemiluminescence Edetic Acid Egtazic Acid Electrophoresis Gels Glycine Immunoglobulins inhibitors leupeptin Milk, Cow's Nitrocellulose Peroxidase Phosphoric Monoester Hydrolases Protease Inhibitors Proteins Sodium Chloride sodium pyrophosphate Tissue, Membrane Tromethamine Tween 20
4
Quantitative Proteomics of Protein Complexes
For immunoprecipitation-western blot experiments, 10 cm plates of 60% confluent HEK293T were transfected with 4 µg of each plasmid (total of 8 µg), and 24 µg PEI. 24 h post transfection, cells were lysed with ice cold IP buffer (10% glycerol, 1% NP-40, 50 mM Tris, pH 7.5, 200 mM NaCl, 2 mM MgCl2) supplemented with protease and phosphatase inhibitor cocktails (539134; MilliporeSigma; BP-479; Boston BioProducts) and incubated on ice for 5 min. Cellular debris was pelleted out by centrifugation at 21,000 g for 15 min at 4°C. Protein concentration was determined using a Bradford assay compared to BSA protein standards, and 1–2 mg of protein was loaded into each IP reaction. Lysates were pre-cleared in 5 µl of magnetic Protein A/G bead slurry (88802; Thermo Fisher Scientific), and 2.5% of the volume of whole cell extract was set aside. Reactions were incubated overnight at 4°C with gentle rotation with mouse monoclonal anti-FLAG (66008-3; ProteinTech Group, RRID:AB_2749837; 5 µg antibody per mg protein). 25 µl magnetic Protein A + G bead slurry was added to each reaction, and incubated for 2 h at 4°C with gentle rotation. Beads were washed three times in IP buffer with 0.1% Tween-20, and proteins were eluted from beads by boiling at 95°C for 10 min in 2X Laemmli buffer diluted in IP buffer. Samples were resolved by SDS-PAGE and Western blotting as described above.
For affinity purification-mass spectrometry experiments, two poly-L-lysine-coated 15 cm plates were each plated with 10 million primary neuron cells. Cells were lysed on D11 and protein samples were handled as described above. Reactions were incubated overnight at 4°C with gentle rotation with mouse control IgG (sc-2025; Santa Cruz) or mouse monoclonal anti-EVL (a gift from Frank Gertler) at 5 µg antibody per mg protein. 25 µl magnetic Protein A/G bead slurry (88802; Thermo Fisher Scientific) was added to each reaction, and incubated for 2 h at 4°C with gentle rotation. Beads were washed three times in IP buffer, and proteins were eluted from beads by boiling at 95°C for 10 min in 2X laemmli buffer diluted in IP buffer. Entire eluate was loaded onto Bolt 4–12% Bis-Tris pre-cast gels (Thermo Fisher Scientific, NW04120BOX), resolved by SDS-PAGE, and the gels were stained with Bio-Safe Coomassie G-250 Stain (#1610786; Bio-Rad).
In-gel digestion: in-gel tryptic digestion was performed as previously described (Kruse et al., 2017 (link)). In brief, each lane of the SDS-PAGE gel was cut into eight slices. Each gel slice was placed in a 0.6 ml LoBind polypropylene tube (Eppendorf), destained twice with 375 µl of 50% acetonitrile (ACN) in 40 mM NH4HCO3 and dehydrated with 100% ACN for 15 min. After removal of the ACN by aspiration, the gel pieces were dried in a vacuum centrifuge at 60°C for 30 min. Trypsin (250 ng; Sigma-Aldrich) in 20 μl of 40 mM NH4HCO3 was added, and the samples were maintained at 4°C for 15 min prior to the addition of 50–100 μl of 40 mM NH4HCO3. The digestion was allowed to proceed at 37°C overnight followed by termination with 10 μl of 5% formic acid (FA). After further incubation at 37°C for 30 min and centrifugation for 1 min, each supernatant was transferred to a clean LoBind polypropylene tube. The extraction procedure was repeated using 40 μl of 0.5% FA, and the two extracts were combined and dried down to ∼5–10 μl followed by the addition of 10 μl 0.05% heptafluorobutyric acid/5% FA (vol/vol) and incubation at room temperature for 15 min. The resulting peptide mixtures were loaded on a solid phase C18 ZipTip (Millipore) and washed with 35 μl 0.005% heptafluorobutyric acid/5% FA (vol/vol) followed by elution first with 4 μl of 50% ACN/1% FA (vol/vol) and then a more stringent elution with 4 μl of 80% ACN/1% FA (vol/vol). The eluates were combined and dried completely by vacuum centrifugation and 6 μl of 0.1% FA (vol/vol) was added followed by sonication for 2 min. 2.5 μl of the final sample was then analyzed by mass spectrometry.
Parker S.S., Ly K.T., Grant A.D., Sweetland J., Wang A.M., Parker J.D., Roman M.R., Saboda K., Roe D.J., Padi M., Wolgemuth C.W., Langlais P, & Mouneimne G. (2023). EVL and MIM/MTSS1 regulate actin cytoskeletal remodeling to promote dendritic filopodia in neurons. The Journal of Cell Biology, 222(5), e202106081.
Publication 2023
acetonitrile Biological Assay Bistris Buffers CD3EAP protein, human Cell Extracts Cells Centrifugation Chromatography, Affinity Cold Temperature Digestion formic acid G-substrate Glycerin Immunoglobulins Immunoprecipitation Laemmli buffer Lysine Magnesium Chloride Mass Spectrometry Mus Neurons Nonidet P-40 Peptide Hydrolases Peptides perfluorobutyric acid Phosphoric Monoester Hydrolases Plasmids Poly A Polypropylenes Proteins SDS-PAGE Sodium Chloride Stains Staphylococcal Protein A Transfection Tromethamine Trypsin Tween 20 Vacuum
5
Western Blot Analysis of Protein Levels
HLE-B3 cells were harvested and lysed using a lysis buffer, and the protein concentration of cell extracts was quantified with a BSA kit. Next, appropriately 20 µg protein was loaded on 10 SDS-PAGE gels and transferred into PVDF membranes, followed by blocking with TBST solution containing 5% skim milk at 4 °C for 3 h. Membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies of Goat Anti-Mouse IgG H&L (HRP) (1:1000, ab205719; Abcam, Cambridge, UK) and Goat Anti-Rabbit IgG H&L (HRP) (1:20000, ab6721; Abcam, Cambridge, UK). Finally, the protein bands were imaged using the Bio-Rad ChemiDoc XRS system. Primary antibodies, including GAPDH (1:2000, 60004-1-Lg; Proteintech, Rosemont, IL, USA), METTL3 (1:1000, 15073-I-AP; Proteintech), and EIF4EBP1 (1:2000, ab32024; Abcam, Cambridge, UK), were used.
Li R., Zhu H., Li Q., Tang J., Jin Y, & Cui H. (2023). METTL3-mediated m6A modification of has_circ_0007905 promotes age-related cataract progression through miR-6749-3p/EIF4EBP1. PeerJ, 11, e14863.
Publication 2023
anti-IgG Antibodies Buffers Cardiac Arrest Cell Extracts GAPDH protein, human Gels Goat METTL3 protein, human Milk, Cow's Mus polyvinylidene fluoride Proteins Rabbits SDS-PAGE Tissue, Membrane

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More about "Cell Extracts"

Cell extracts, also known as cellular lysates or subcellular fractions, are highly versatile tools in biochemistry and molecular biology research.
These preparations, obtained by disrupting or lysing cells, contain a wealth of intracellular contents, including organelles, proteins, nucleic acids, and other biomolecules.
The composition and properties of cell extracts can be optimized through careful selection of extraction methods and conditions, such as the use of PVDF membranes, protease inhibitor cocktails, and techniques like the Dual-Luciferase Reporter Assay System.
Cell extracts are widely employed to study a range of cellular processes, including protein interactions, enzyme activities, and signaling pathways.
Researchers often rely on cell extracts to conduct experiments involving β-actin, a commonly used reference protein, and utilize detection methods like the Pierce BCA Protein Assay Kit and the Odyssey Infrared Imaging System to quantify and analyze the extracted biomolecules.
The accuracy and reproducibility of cell extract-based experiments are of utmost importance, and PubCompare.ai's AI-driven platform helps researchers identify the most effective protocols from literature, preprints, and patents.
By leveraging cutting-edge comparisons, the platform can pinpoint the most accurate and reproducible cell extract preparation techniques, such as those involving Lipofectamine 2000 and Complete protease inhibitor cocktail, to enhance the quality and reliability of your research.
With the power of AI at your fingertips, you can take your cell extract studies to new heights, unlocking new insights and advancing scientific knowledge.