WT Dictyostelium (strain AX3 and AX2), adenylyl cyclase A null cells (aca−) [14] (link), myosin-II null cells (myoII−) [15] (link) and talin A null cells (talin A−) [12] (link) were grown in HL-5 medium to 4–5×106 cells/ml. For all experiments, cells were developed for 4.5 (WT) or 5 (aca−, myoII−, talin A−) hrs in development buffer (DB; 5 mM Na2HPO4, 5 mM NaH2PO4, pH 6.2, 2 mM MgSO4 and 0.2 mM CaCl2) at 2×107 cells/ml, with exogenous pulses of 75 nM cAMP every 6 min, as previously described [16] (link). Unless otherwise noted, cells were taken from development or growth and centrifuged at 500 g for 3 min. The supernatant was aspirated and the pellet was washed twice with phosphate buffer (PB; 5 mM Na2HPO4, 5 mM NaH2PO4, pH 6.2) and finally resuspended in PB and processed according to the assay performed. Experiments requiring Latrunculin A treatment involved resuspending cells in PB with 5 µM Latrunculin A (Invitrogen) for 5 min before placing them in a chamber in which the buffer contained 5 µM Latrunculin A. For the Latrunculin A titration experiments, cells were resuspended in PB with different concentration of Latrunculin A (0 (only PB), 5 µM, 1.5 µM, 0.5 µM, 0.25 µM) for 5 min before placing them in a chamber where the buffer contained the same concentration of Latrunculin A.
Latrunculin a
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan
Latrunculin A is a small molecule that has the ability to disrupt the actin cytoskeleton of cells. It binds to actin monomers, preventing their polymerization and thereby disrupting the dynamic structure of the actin filaments.
Automatically generated - may contain errors
Lab products found in correlation
Jasplakinolide, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 555 phalloidin, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Cellmask, by Thermo Fisher Scientific (2 mentions)
Benomyl, by Merck Group (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Nis elements software, by Nikon (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Fluorodish, by World Precision Instruments (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Syk inhibitor r406, by Selleck Chemicals (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Anti β catenin, by Thermo Fisher Scientific (1 mentions)
Plan apo 60x, by Nikon (1 mentions)
Nb100 1614, by Novus Biologicals (1 mentions)
Nocodazole, by Thermo Fisher Scientific (1 mentions)
Double thymidine, by Merck Group (1 mentions)
Pertussis toxin, by Merck Group (1 mentions)
Hiperfect, by Qiagen (1 mentions)
36 protocols using latrunculin a
1
Dictyostelium Cell Development Assays
2
Investigating Synaptic Vesicle Dynamics in HD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study any rescue effect of jasplakinolide on the dynamics of non-releasing synaptic vesicles in HD neurons, jasplakinolide (5 μM; J7473, Thermo Fisher Scientific) or vehicle (DMSO) was added to the chamber after the addition of trypan blue to the extracellular solution. Imaging experiments were performed 10 min later as described in the experimental method section for real-time imaging of single QD-labeled synaptic vesicles in this paper. To study any effect of Latrunculin A on the dynamics of non-releasing synaptic vesicles, 0.1 μM Latrunculin A (L12370, Thermo Fisher Scientific) or vehicle (DMSO) was added to the chamber through perfusion system after trypan blue perfusion. After 3 min perfusion of Latrunculin A, imaging experiments were performed as described in the experimental method section for real-time imaging of single QD-labeled synaptic vesicles.
3
Immobilizing Sporulating Yeast Cells for Live Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After overnight incubation on ME plates, cells were re-suspended in EMM-N+5S medium. To disperse sporulating cells, suspensions were subjected by brief sonication (Handy Sonic; Tomy Seiko, Tokyo, Japan); 20 µl of the cell suspension was then dropped onto lectin (0.2 mg/ml; Sigma-Aldrich, Tokyo, Japan)-coated 35-mm glass-bottomed culture dishes (MatTek, Ashland, MA, USA) to immobilize cells (Asakawa and Hiraoka, 2009 (link)). For imaging Latrunculin A-treated cells, Latrunculin A (Thermo Fisher Scientific, Tokyo, Japan) was added at a final concentration of 1 µM prior to cell immobilization. Cells undergoing sporulation were selected for live-cell imaging.
A DeltaVision microscope equipped with a CoolSNAP HQ2 charge-coupled device (GE Healthcare, Tokyo, Japan) was used for image acquisition. Optical section images were acquired at 0.5-µm focus intervals using an oil-immersion 60× objective lens (PlanApoN60x OSC, NA1.4; Olympus, Tokyo, Japan). Images were processed using the de-noising algorithm (Boulanger et al., 2009 (link)) and by constrained iterative deconvolution (Agard et al., 1989 (link)).
A DeltaVision microscope equipped with a CoolSNAP HQ2 charge-coupled device (GE Healthcare, Tokyo, Japan) was used for image acquisition. Optical section images were acquired at 0.5-µm focus intervals using an oil-immersion 60× objective lens (PlanApoN60x OSC, NA1.4; Olympus, Tokyo, Japan). Images were processed using the de-noising algorithm (Boulanger et al., 2009 (link)) and by constrained iterative deconvolution (Agard et al., 1989 (link)).
4
Immunofluorescence Staining of Cell-Cell Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunofluorescence, cells were fixed and permeabilized either with methanol-acetone or, in case of phalloidin staining, with 3% formaldehyde–1% Triton X-100, or, in case of anti-vinculin staining, with BM[PEO]3 (see Indra et al., 2013 (link) for details). Wide-field images were taken using a microscope (Plan Apochromat 100×/1.40 NA objective lens; Eclipse 80i; Nikon) and a digital camera (CoolSNAP EZ; Photometrics). The images were then processed using NIS-Elements software (Nikon). The following antibodies were used: mouse anti–E-cadherin and anti-occludin (Invitrogen), rabbit anti-Dendra2 (Evrogen), mouse anti–β-catenin and ZO1 (BD), mouse anti-vinculin and rabbit anti-EPLIN (Sigma-Aldrich), goat anti–α-catenin (Santa Cruz Biotechnology, Inc.), rabbit anti–α-catenin N-terminal domain, EP1993Y (Abcam), and guinea pig anti-cingulin (provided by I. Hofmann, German Cancer Research Center, Heidelberg, Germany). Alexa Fluor 555 phalloidin and Latrunculin A were purchased from Invitrogen.
5
EDTA-Induced Cell Rounding Experiments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For EDTA-induced cell rounding experiments (Fig. 2, D–F ), the medium was first replaced with PBS at least 20 min before adding 1 mM EDTA in PBS. PBS and PBS-EDTA have equal osmolarity. For experiments with prerounded HeLa cells (Fig. 2, A–C ), cells were prerounded by placing them in chambers prepared as previously described but coated with PLL-g-PEG (1%) instead of fibronectin after plasma treatment.
Experiments with latrunculin A from Invitrogen and Y27632 from Calbiochem were performed in chambers preincubated with the final concentration of drug one night prior seeding the cells to prevent drug depletion caused by hydrophobicity or PDMS. Images of the cells were taken before launching acquisition to confirm the effect of the drug (Fig. 3 A ).
Experiments with latrunculin A from Invitrogen and Y27632 from Calbiochem were performed in chambers preincubated with the final concentration of drug one night prior seeding the cells to prevent drug depletion caused by hydrophobicity or PDMS. Images of the cells were taken before launching acquisition to confirm the effect of the drug (
6
Actin Cytoskeleton Disruption Assay
7
Cell Culture and AFM Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH 3T3 and MDA-MB-231 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1% antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA) in a humidified 5% CO2 atmosphere at 37 °C. Prior to AFM experiments, cells were re-plated onto 50 mm glass-bottom cell culture dishes (FluoroDish, World Precision Instruments, Sarasota, FL) coated with 30 μg/mL fibronectin for 30 min (Sigma-Aldrich, St. Louis, MO) and grown for an additional period of 1−2 days to a final confluency of ~60–70%. F-actin depolymerizing drug Latrunculin A (Invitrogen, Carlsbad, CA) was applied at a concentration of 100 nM from the DMSO stock solution.
8
Visualizing Actin Dynamics and Signaling in Dictyostelium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells of D. discoideum strain AX2-214 expressed mRFP-LimEΔ or LimE∆-GFP [72 (link),73 (link)] as an actin probe together with either an mRFP-Raf1-RBD construct, the minimal Ras-binding domain (RBD) of human Raf proto-oncogene serine/threonine-protein kinase (Raf-1) [74 (link)], superfolding-GFP-PHcrac [75 ] as a label for PIP3, coronin-GFP, mRFP-coronin [42 (link)], or GFP-myosin IB [39 (link)]. Expression of PTEN-GFP [76 (link)], a label for PI3-phosphatase was combined with mRFP-Raf1-RBD, and GFP-myosin IB with mRFP-coronin. PTEN-null cells [77 (link),78 (link)] were transfected with mRFP-LimEΔ together with superfolding-GFP-PHcrac or GFP-Raf1-RBD.
The cells were cultivated in Petri dishes with nutrient medium containing 10 µg/ml of blasticidin (Invitrogen, Life Technologies, Grand Island, NY, USA) and 10 µg/ml of G418 (Sigma-Aldrich, St. Louis, Missouri, USA). They were harvested from the Petri dishes and washed twice in 17 mM K/Na-phosphate buffer pH 6.0 (PB). An aliquot was pipetted in a HCl-cleaned cover-glass bottom dish (FluoroDish, WPI INC., Sarasota, FL, USA) with PB for TIRF imaging. Waves were initiated either by adding 5 µM Latrunculin A (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and (except forFigure 3 ) exchanging it for PB after 15 min, or they were spontaneously formed in large cells produced by electric-pulse induced fusion [53 (link)].
The cells were cultivated in Petri dishes with nutrient medium containing 10 µg/ml of blasticidin (Invitrogen, Life Technologies, Grand Island, NY, USA) and 10 µg/ml of G418 (Sigma-Aldrich, St. Louis, Missouri, USA). They were harvested from the Petri dishes and washed twice in 17 mM K/Na-phosphate buffer pH 6.0 (PB). An aliquot was pipetted in a HCl-cleaned cover-glass bottom dish (FluoroDish, WPI INC., Sarasota, FL, USA) with PB for TIRF imaging. Waves were initiated either by adding 5 µM Latrunculin A (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and (except for
9
Visualizing PTEN-Halo Dynamics in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To label wild type PTEN-Halo or PTENi-Halo, prepared cells were incubated with DB including 100 nM TMR conjugated HaloTag ligands for 10 minutes. The cells were then washed and suspended in DB and placed on a glass cover slip. After 10 minutes, cells were overlaid with a sheet of agarose and incubated for 20 minutes [29] (link). To inhibit actin polymerization, cells were treated with 10 µM Latrunculin A (Invitrogen). The sheet of agarose was also treated with 10 µM Latrunculin A before use. Single molecules of wild type PTEN-Halo and PTENi-Halo were visualized through objective type TIRFM. Details of the TIRFM configuration are described elsewhere [23] (link).
10
Contextual Fear Conditioning in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All experiments were performed with 2-month-old male mice. The cFC training context was rectangular in shape. Identity of the context was maintained with the presence of distinct odor (2% acetic acid, v/v). The conditioning chamber was cleaned with 70% ethanol before and after each session. Mice were single housed and were first handled for 5 min for 3 d. On training day, mice were allowed to explore the training context for 1 min and then received 3 foot shocks (2 s and 0.6 mA each, intertrial interval 30 s). Contextual fear memory was assessed by returning mice to the training context 24 h after fear conditioning and analyzing freezing during a test period of 2 min. Freezing was defined as complete absence of somatic mobility other than respiratory movements. No animals were excluded from the analysis. In some experiments, jasplakinolide or latrunculin A (Invitrogen) was freshly dissolved in DMSO (3% in normal saline; 100 μl) and then injected intrathecally at a dose of 0.5 μg/mice immediately after training.
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!