... 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 ...

... This is an important downside of the technology and should always be considered when interpreting results...oligomerization of fluorescent proteins is a concern. The propensity of fluorescent proteins to oligomerize is known...brightness, photostability, maturation time, and acid sensitivity of various fluorescent proteins (Botman et al...coded by the multiple cloning site and therefore consist of random amino acid sequences or (ii) designed... an inert, unstructured peptide and therefore consisted of glycines, serines and other small, non-aliphatic...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...fluorescent protein fusions There are several choices to consider before generating a fusion protein, e.g. linker...

... other ligands - almost A to Z! - from ATP to urotensin. The MTRIA sensors will be useful for investigating...displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR. Image from... response kinetics and increased brightness, sensitivity, and photostability under two-photon illumination...proteins (miRFP670nano and miRFP720) and the Ca2+-sensing domain from Twitch-2B (Matlashov et al. 2022). ...plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium ...LiLac has improved response size and reduced sensitivity to pH and calcium compared to previous lactate...470-495 nm) and is capable of local temperature sensing with sub-millisecond resolution and sub-degree ...

... Morgridge Institute for Research in Madison, Wisconsin. It is commonly cited that approximately one-third...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...The development of phosphoserine incorporation Extensive genomic sequencing of prokaryotic lineages revealed...specialized elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide...available through Addgene, but we have worked most extensively worked with and optimized Sep-OTSλ and can recommend...the deletion of release factor 1, the protein responsible for terminating translation in response to the...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...

...integrating a SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore...organoids By Ashley Waldron Organoids are three-dimensional cultures that mimic aspects of organ morphology...enzyme biosensors provide direct, specific and sensitive detection of the plant hormones strigolactones...the receptor which modulates the fluorescence intensity of the cpGFP without altering that of the internal...fluorophores, rather than on the fluorescence intensity, it provides a direct and quantitative detection...receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for...

...temperature drops, single intensity should smoothly increase as the DNA amplicon transitions from single-stranded...detect a single copy of a transcript. But this sensitivity can sometimes cause as many issues as it solves...separate thermocycle allows you to measure the intensity of signal over an entire cycle. Since signals ...double-stranded DNA, when temperatures are high, signal intensity should be low, reflecting the presence of primarily...assumption that there is a smooth, single-step transition between ssDNA and dsDNA. This is not true for...one (Biassoni, 2014). At the bench As a highly sensitive technique, qPCR needs a steady pair of hands to...important as precision for relative quantification. A consistent 5% positive error across the plate is fine; a...

...should be consistent across all samples on the gel and should be observed as a band of consistent size and...These systems, however, require you to purchase expensive system-specific sandwiches.   Figure 2:...vendors will show that the antibody binds to transiently overexpressed protein, however, they should also...includes publications that cited the antibody, consider reviewing the materials and methods sections of...and density present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent...you require precise quantification of a protein, consider using an alternative method such as an enzyme-...

...ultimately responsible for outcomes. Be ready to support your team and take responsibility if things go...series on Management for scientists. Once you are responsible for managing others you will only be successful...you become a master at delegating tasks and responsibilities. The manager is not expected to DO all the...experience? Do you think you are giving this responsibility to someone who can do the job. It is good to...sure it goes well? As with most managerial responsibilities, good communication is at the heart of delegating...boundaries. Where are the lines of authority, responsibility and accountability? What types of issues do...

...resultant cells can either express the plasmid DNA transiently (similar to bacteria) or incorporate the genetic...ever-versatile fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For... for the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection...want the plasmid depleted, but still want to use transient transfection methods. Since there are no "natural... generations. Stably transfected cells may be considered an entirely new cell line from that of the original...plasmid contains a cassette that will make cells sensitive to a toxin). In the table below we focus on positive...causes strand scissions 100-400 ug/mL *Not comprehensive.   ** In eukaryotes.   ***The concentration ...