HeLa cells expressing either control or cenexin shRNA (Hung et al., 2016 (link)) were transfected with PLK1 FRET-PACT sensor using Mirus Bio TransIT-X transfection reagent. After 48 h, cells were treated with 100 nM nocodazole and synchronously released into metaphase after 6 h to induce lagging chromosomes. Cells were imaged live or then fixed using 4% PFA containing 0.5% Triton X-100 at room temperature for 30 min. Cells were immunostained for centrobin (1:500) or centrin (1:1000) to distinguish between the spindle poles and CREST (1:1000) to visualize kinetochores. Coverslips were then mounted in vectashield and imaged using a Leica DMi8 STP800 (Leica, Bannockburn, IL) equipped with an 89 North–LDI laser, Photometrics Prime-95B camera, Crest Optics: X-light V2 Confocal Unit spinning disk, using a HC PL APO 63×/1.40 NA oil CS2 objective. For these experiments, YFPex→YFPem control images were taken, using a 520-excitation laser. CFPex→YFPem was imaged using a 445-excitation laser. The YFPex→YFPem/CFPex→YFPem FRET ratio was calculated using ImageJ Ratio-Plus plug-in after background subtraction and averaged over multiple cells. Experiments were repeated multiple times with similar results.
Transit x transfection reagent
Manufactured by Mirus Bio
The TransIT-X transfection reagent is a versatile and efficient tool for transfecting a wide range of cell types. It facilitates the delivery of nucleic acids, such as plasmid DNA, mRNA, and siRNA, into cells, enabling effective gene expression or gene silencing studies.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using transit x transfection reagent
1
Induction of Lagging Chromosomes in HeLa Cells
2
Measuring Cell-Cell Fusion Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The HEK293T cells were plated out to a cell density of 7.5 × 105 cells per well in 6 well dishes. The following day, effector cells were transfected with 500 ng each of MeV or PPRV F and H expression constructs, 500 ng of the 1–7 fragment of rLuc-GFP [25 (link)] and 1 μg of either the blank vector control (pcDNA3.1) or a BST2 expression vector (as indicated). Separately, target cells were transfected with 1 μg of lentivirus vectors expressing human or ovine SLAMF1, as well as 500 ng of the 8–11 fragment of rLuc-GFP. All transfections were performed using TransitX transfection reagent (Mirus), according to the manufacturer’s instructions. Following 48 h of incubation, effector and target cells were washed, counted, and co-cultured at a ratio of 1:1 in white-walled 96 well plates to a final density of 1 × 105 cells per well. Then, 16–24 h later the Renilla luciferase activity in fused cells was measured (in a Promega GloMax multi-mode plate reader) by removing the media and adding 2 μg/mL of cell-permeable coelenterazine 400A (Biotium, Fremont, CA, USA), in PBS. Normally, five or more co-culturing replicates were performed for each biological condition.
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!