...proteins in cells. Homeostatic regulation of protein abundance is essential for normal cell function, and...one particular protein in neurons or in any other cell type. However, we cannot distinguish new from old...facilitate the visualization of specific proteins in cells. Tagged protein can easily be overexpressed from...techniques to follow translation in living single cells. However, before CRISPR-Cas9 these techniques were...interests, suspected to interact, are added to a cellular sample. Both antibodies have a short antibody ... turnover of specific endogenous proteins in any cell type. Exciting! Many thanks to our guest blogger... how small molecule fluorophores can be used for cell labeling Check out other protein tagging strategies...

.... RANbodies are expressed in 293T cells and are secreted from cells due to their IgGκ signal peptide sequence...-to tool for detecting a protein of interest in cells and tissues. Although antibody production is well... sequence, making them easy to harvest from cell culture media. Their His tags allow for further purification...RANBody While all three reporters stain cultured cells and tissue sections, some reporters are better suited... signal amplification of HRP Staining cultured cells and tissue sections Amplifying weak signals of fluorescent...Labeled anti-HA or -Myc antibody Staining cultured cells and tissue sections When detected with a labeled...Labeled Anti-Chicken IgY antibody Staining cultured cells and tissue sections Great for multicolor staining...

...sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses on the enzyme hydropersulfide-... understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: Micrographs...Micrographs of the biosensor expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP...studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute to the ...will explore the connectivity patterns of these cells and the functional contribution to a variety of ...administration and withdrawal to explore how these cells may encode the negative affect associated with drug...library of mutant sensors expressed in a mammalian cell line engineered to be electrically excitable. The...

...Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System.” Cell 155 (2013): 1379-1491. Pubmed...of 6 chromosome-specific loci in the U20S cancer cell line./ Each color represents a gRNA targeting a ...localization and movement of this locus in live cells! This technique was developed in the lab of Addgene...et al. to track telomere dynamics throughout the cell cycle. To optimize the signal to noise ratio of ...sophisticated tracking of multiple chromosomal loci in live cells. However, they note that certain improvements could...repression and genome editing. Ready to make your cells a little more colorful? CRISPRainbow is available...Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. USA 112(10) (2015): 3002...

... yeast, plant, insect, and mammalian cells, including human cells. piggyBac is biased to integrate at ...is active in vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type piggyBac...G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo...and has lower efficiency of integration in human cells than piggyBac and Sleeping Beauty. Minimal Tol2 ... or barcodes. When transposons are delivered to cells or a model organism, they insert into the host’s...

...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Yan, J., Oyler-Castrillo...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment. Now, onto the ...nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...also allows certain unintended edits to escape cellular surveillance. To limit unintended incorporation...https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A...

...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...allow you to study natural genetic circuits in the cell. Natural and synthetic product production A major...biofuels, drug precursors, etc., microbially or in cell-free systems. These pathways typically involve multiple...

...CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed...RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227.   Potential ...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. What we still don't know about Cpf1...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016 Jun 6. PubMed PMID: 27272384...specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology (2016). PubMed PMID: 27347757...

... primary cells. The authors used this platform to assay dozens of GPCRs across many cell types in...biosensor platform. Cell, 187(6), 1527–1546.e25. doi: https://doi.org/10.1016/j.cell.2024.01.028. New...Prime editing (PE), initially developed in human cells, has been uniquely challenging to apply in dicotyledonous...labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then used single-molecule..., CLIPf-, and CLAP-tagged proteins for mammalian cell expression. These plasmids will be useful not only...recordings of spontaneous activity in human stem cell-derived neurons expressing ASAP5-Kv. C) Cultured...

...high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (<15 μm) tissue...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter whether...1). Thin dynamic samples Ex: monolayers of live cells with fluorescently labelled, motile structures These...Linear Fluorescence Transitions (RESOLFT/STED) also excel on these types of samples. Prior to the advent of... 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, small model embryos >15 μm Thsee are some...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional ...Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol 361, 1-33 (2003). PubMed...

...opsin expression in the intended brain region and/or cell type (I’ll talk more about this in detail later)...desired optogenetic construct in the brain region and cell type of interest is obviously of critical importance...wish to stimulate or inhibit neuronal firing, the cell-type specificity, the kinetics of the opsin variant...control of a generic promoter). A DIO opsin allows cell type specificity dependent on the promoter of the...expression of your opsin is no longer required to achieve cell-type specific opsin expression. However, the types...spread, and with co-localization immunofluorescence, cell-type specificity. Viruses that only express the ...experimentation.  Whether you attempt to stimulate the cell body or an axon terminal also affects the length...

...produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells are fused with activated...activated B cells from the spleen of mice injected with the antigen of interest. The immortalized cells, called...distributing over 3,000 antibodies and associated hybridoma cell lines. The DSHB was created by the National Institute... individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing...

...both Cas9 and your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer...knock-out thousands of genes in mixed population of cells as part of pooled library screens (1,2). The human...pooled libraries targeting both mouse and human cells are avaliable for distribution. To learn more about... includes work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase...of the many plasmids being distributed to and accelerating research in labs all around the world. We thank...Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87. PubMed PMID: 24336571...Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit our Lentiviral...

...both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus...the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of ... puromycin resistance gene enables selection of cells with the plasmid post transfection. The 2A "self...can be used to transfect a variety of mammalian cells and can be modified for viral trasduction (see the...resistance gene that made it less effective in certain cell lines. This point mutation has been corrected in... your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain ...Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit our CRISPR...

... Guide RNA and Target DNA. Cell 156:935–949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong ... Delivery by AAV Vectors. Cell 181:136–150 . https://doi.org/10.1016/j.cell.2020.03.023 Additional Resources...acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels at a similar efficiency ...package each piece into an individual AAV. When a cell is transduced by both AAVs, the full length gene...SpCas9 which had modest editing rates in HEK-293T cells when compared to the full-length SpCas9. In 2016... allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: ....

..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting temperature...cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further enhance...background in vivo. The nature of aptamers makes them excellent candidates for developing assays for otherwise...Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. USA 112(10) (2015): 3002...

...if the cell dies from injury, this protein leaks out into the cytoplasm, migrates out of the cell, and ...Keytruda) causes T-cells to more easily recognize and cause the destruction of cancer cells. Pembrolizumab...cause inflammation, which can cause mesothelial cells to mutate and ultimately form cancerous tumors.  ...resection), systemic chemotherapy to kill cancerous cells that may have spread beyond the point of origin,...successful in killing many (if not all) of the cancer cells that may be left behind after surgery, while being...and binds cells that can begin an immune response. This important biomarker can be used to distinguish between...

...(FRET) microscopy imaging of live cell protein localizations. J Cell Biol. 2003 Mar 3;160(5):629-33. ...measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions...suitable for studying interactions within living cells. By coupling fluorophores to proteins, pioneers ...directly detect protein/protein interactions in living cells. Since then FRET has also been used to measure conformational... Constructs to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian...efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies Several methods have been used over...microscopy: monitoring protein interactions in living cells. Bioessays. 2012 May; 34(5):341-50. doi: 10.1002...

... Ba/F3 and MCF10A cellular models. Ba/F3 and MCF10A are growth factor dependent cell lines and any mutations...attempting to characterize mutations in a pooled cell population, as mutations with strong phenotypic ...functional groups, based on how each one impacted cell viability (see figure). Most of the mutations were...to be “neutral,” having no significant effect on cell viability compared to a wild-type control. Approximately... the mutations, however, significantly increased cell viability, and these “activators” may be the direct...annotation of somatic mutations in cancer." Cancer Cell 33.3 (2018): 450-462. PubMed PMID: 29533785. PubMed...

... are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct...shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research...turnover rate. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would...made by accident. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. ...spontaneous conversion did not occur. Tobacco suspension cell culture with cytosol-targeted GFP. White circles...this technique may not be appropriate for high-pH cellular compartments. Sattarzadeh et al. have shown that...photoconverted in plant, Drosophila, and mammalian cells. Photoconverted GFP is compatible with other FPs...