...specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the ...thus tracking the localization of that biomolecule inside living cells. FPs classified by the emission color...range, there are other traits that need to be considered when choosing an FP: Unique categories of fluorescent...distance between the two FPs is <10nm, and should be considered when labeling proteins that interact. Indeed,...Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For instance...experimental parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent...experimental parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter...

...biologists were no longer beholden to chemists and (expensive) synthetic fluorophores. Add a dash of DNA with...to small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances...requires more photons. For example, moving from transient overexpression of protein fusions to gene-edited... HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that...fluorogens (Figure 1e). Stains – These labels consist of a fluorophores conjugated to molecular species...can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling tag systems...groups (Figure 2b), which can preclude their use inside living cells. We recently discovered that incorporation...

...tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2...dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids created for ...plasmid backbone that was designed specifically for transient expression (find some useful backbones here) or... or from viral transduction. While not ideal, transient expression of viral constructs is a useful tool...cell line. When expressing a viral construct transiently care needs to be taken to ensure that the proper...differ in two ways. Firstly, 2nd generation systems consist of an envelope plasmid and a packaging plasmid ... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...

...come up with a list of things to consider as you make this transition. Of course, a “desk job” could entail...employee who's coming directly from grad school. The transition has been a bit rough for her (3 weeks in and ...there are any resources that can help with the transition.” The lab-to-desk-job culture shock is a real...scientist colleagues have as well. Making the transition from the bench can be jarring for the unprepared...resources out there for scientists making this transition. In this installment of the Addgene career guide...What should you expect during the lab → office transition? I’ve already provided a few counterpoints to...likely happen eventually. If you approach that transition dreading the change, the adjustment period will...

...develops an enhanced propensity for movement and activity called locomotor sensitization. This enhanced movement... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...ve previously discussed some of the general considerations for performing behavioral tests, but now we...seeking drugs’).  What’s involved in locomotor sensitization? Previous research has shown that regions such... glutamate transmission leads to locomotion sensitization. (for review see van Huijstee & Mansvelder  ...suggested inhibitory mechanism of locomotor sensitization and might open new ways for a novel therapeutic...subset of excitatory neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically...

...training schedule that helped me transition easily. Training and responsibilities Through this training, I was...The application process When I first saw the comprehensive job description for this co-op, I was immediately...me a full description of the tasks I would be responsible for, in comparison to the other positions listed...for the independent work on the QC team. I was responsible for helping other scientists on the team ensure...sequencing QC work    In addition to my responsibilities on the QC team, I was able to explore other...uses something called “core hours”. Core hours consist of the time between 10 AM and 3 PM, when most meetings... the “9-5” was not as scary as it seemed. The transition into a traditional work environment was aided...

...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers... with fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks...Optogenetics functions through opsins which are light sensitive proteins. Opsins used in optogenetic studies are...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...inhibition: Hyperpolarization Conversely, when the inside of the neuron becomes more negative it becomes ...differing peak absorption wavelengths and light intensity requirements of different opsins, studies have...

...A major limitation of APEX was its low sensitivity. Sensitivity could be increased with higher expression... avidin, proximity labelling can capture weak/transient protein interactions as well as be used for both...promiscuous biotin ligase (Kim et al 2016). BioID2 consists of a humanized biotin ligase from A. aeolicus,...cell.   Find BioID2 plasmids here APEX2: A more sensitive ascorbate peroxidase for proximity labeling A ...aggregation. The Ting lab hypothesized that the low sensitivity was due to suboptimal folding or stability. Thus...identified a mutant with significantly increased sensitivity that did not affect the function or efficiency... assemble when in close proximity. Split-BioID consists of a split BirA* molecule that can reassemble ...

... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...histogram, which graphically represents the time and intensity of the cell’s illumination (see Figure 2). H describes...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...as our cells of interest do not lie beyond the dimensions displayed in the plot. (B) A more restrictive... marker stains of interest). Let’s double-click inside our gate and proceed to the next plot! Pro tip!... on to my experimentally-specific gates. Two-dimensional staining (quadrant gating) For our last example...

... are considering a career at a small, teaching college you might want to consider less expensive, portable...low salary I might add). You have a right and responsibility to choose a lab where you can thrive and do...Former lab members can give you a fuller picture. Consider talking to people in the lab across the hall or... hope to grow.  Find mentors who take their responsibility to train you seriously. Why work for someone...whether or not a lab head is a good role model, consider questions like: How long do lab members stay ...allocation of meaningless tasks, removal of responsibility, repeated reminders of blunders, setting target...equipment quotes? Understand administrative responsibilities? Is the work (topic and/or technical aspects...

... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...your experiment, you might need to quantify the intensity of the different signals in a certain region or...optimizing your blocking and washing conditions or consider using a different antibody.  Prepare a positive...

...variation between iPSC clones. Retroviruses are not considered safe for clinical applications.     Lentiviral...Like retroviruses, lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox...same as using a standard lentiviral vector, but transient expression of Cre recombinase allows for deletion... site of excision, this approach is still not considered safe for therapeutic applications. piggyBac (...insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors ...many passages. Ban et al developed a temperature sensitive Sendai virus which is removed by culturing cells... regulate Sendai virus transgene expression. Transient episomal delivery Non-integrating methods have...

...capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—...between male and female cells. The result of not considering the sex of the stem cells was that their male... in Norway, the team realized they should also consider the sex of the stem cells. In this case, they ... many researchers I’ve talked to who’ve never considered the sex of their cells. For one thing, cells ... there are numerous methodological articles to consider—we have loaded those we are aware of in our checklist...clue or potential place to start research is by considering sex disparities in disease severity. Such disparities...checklist. A common worry for researchers wanting to consider sex is cost. Especially in animal research, they...

...Cleavage Assay Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided...inappropriate for heterogenous populations, low sensitivity Targeted Amplicon Sequencing Provided 0.01%...0.01% (variable) Sequence-level data, extremely sensitive Do not sequence all DSBs, may miss unpredicted ...information from targeted amplicon sequencing is highly sensitive with detection levels as low as .01% (Hendel et... with in silico prediction to create a more comprehensive list of off-target editing events. Two of the...laboratories. Nevertheless, unbiased detection isn’t as sensitive as targeted amplicon sequencing. For example, ...throughout the genome. However, their decreased sensitivity means that the best option moving forward may...

...available with improved kinetic and sensitivity parameters. Sensitivity and Localization The calcium concentrations...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...kinetics: GCaMPf (fast) GCaMPm (medium), GCaMPs (sensitive but slower). GCaMPf may be suitable for very rapid...You want to use a calcium sensor that is most sensitive to your expected range. For example, calcium in... changes with Ca while another wavelength is insensitive. Additional red sensors based on mRuby (jRCaMP1...unnecessary calcium buffering and to increase sensitivity), and is a great FRET sensor option if you have...integration within the target cells, ensuring more consistent and reproducible results in calcium assays. There...

...and potential delivery methods.   General considerations for planning your CRISPR experiment As with...experiment, there are many factors that need to be considered during the planning process. For CRISPR experiments... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...are compatible with all your components. Next, consider the best form for those CRISPR components based...selection marker to generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a ...transfer vector and used to generate AAV particles. Transient or stable expression of SaCas9 and/or gRNA. Infects...cells using microinjection or electroporation. Transient expression of CRISPR components, expression decreases...

...showed greater photostability and also matured considerably faster, with expression levels comparable to...PILATeS, Pulling-Induced Luciferase Association Tension Sensor (Zhong et al., 2024). In this new variation...and only exposed when the module unfolds under tension. When this happens, BiT and the LgBiT luciferase... Figure 5: Left: Mechanism of the PILATeS tension sensor. The LgBiT (cyan) fragment of luciferase...protein (purple) only when Titin I10 unfolds under tension. Upon binding, LgBiT and BiT Tag catalyze a luminescent...PILATeS-functionalized coverslip creates mechanical tension, and the luminescence colocalizes with the focal...lower background signal and can be used in light-sensitive systems. Most excitingly, assembly of the split...

...first-generation APEX suffered from inconsistent activity levels and limited sensitivity under the stringently low...extremely dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP...GFPs with diversity in fluorescence brightness, intensity, excitation and emission spectra. The lab of Dimitri... degradation. Chemical approaches may require expensive peptides or small molecules and exceed a physiologically...physiologically relevant exposure in intensity and/or duration. Both of these strategies carry the risk of...activate signaling cascades on exposure to low-intensity blue light. This approach relies on the incorporation...incorporation of light-oxygen-voltage (LOV) sensing domains from algae into chimeric Opto-RTKs. The LOV domains...

...brain. Sensitivity In ex vivo studies on mouse brain slices, nLightG and GRABNE were more sensitive than...But this first generation of tools had limited sensitivity and speed and were only available as green probes... vivo experiments. nLight indicators are more sensitive to and selective for norepinephrine and faster...that red fluorescent proteins tend to be less sensitive than green fluorescent proteins. nLightR did perform...and may soon have a red tool which matches the sensitivity of nLightG. Many thanks to Mike Waring and Tommaso...Bohacek, J., Reiner, A., … Patriarchi, T. (2023). Sensitive multicolor indicators for monitoring norepinephrine...

...antibodies when possible. If cost is prohibitive, consider an approach that prioritizes more reproducible...with representative images showing the staining intensity in the KO population (pink histogram) compared...knockout experiments can be time-consuming and expensive, it may be helpful to find an antibody that has...RNA (siRNA). Knockdown caused by siRNA is also transient, and as such, the optimal knockdown of the protein... rate. If you need a more permanent knockdown, consider using shRNA vectors instead. With both siRNA and...understand the relationship between antibody staining intensity by flow cytometry, and mRNA expression.   ... at similar levels in your cell lines, you can consider a cell treatment approach. If there is a cell ...