Wild-type and adcB−C− cells expressing cAR1-YFP or cAR1cm1234-YFP were plated in four-well chambers (Nalge Nunc International) in 400 μl of DB. The cells were stimulated at the indicated dose of cAMP by applying a 100-μl volume of cAMP into the well. The internalization of cAR1-YFP was imaged using a Zeiss 710 or 780 LSM confocal microscope using an EC plan-Neofluar 40× oil lens. Photographs were taken after 1 h. The internalization was assessed by normalizing the intensity of YFP in the cytoplasm to the membrane of the cells.
Four well chamber
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Four-well chambers are a type of lab equipment designed to hold and contain multiple samples or specimens during various experimental procedures. Each chamber has four individual wells, allowing for the simultaneous processing or observation of up to four separate samples. The core function of these chambers is to provide a controlled and isolated environment for the samples, facilitating consistent and reliable experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
710 confocal microscope, by Zeiss (1 mentions)
Blocking buffer, by Cell Signaling Technology (1 mentions)
P mlc2 antibody, by Cell Signaling Technology (1 mentions)
Lsm900 scanning microscope, by Zeiss (1 mentions)
Zen 3, by Zeiss (1 mentions)
Acti stain 555 phalloidin, by Cytoskeleton (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
4 protocols using four well chamber
1
Imaging cAR1 Internalization in Dictyostelium
2
Visualizing Membrane Recruitment of AdcC
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Cells expressing AdcC-YFP or AdcC-mCherry were plated in four-well chambers (Nalge Nunc International, Naperville, IL) in 400 μl of DB and were stimulated with 10 μM cAMP by applying a 100-μl volume of cAMP into the well as described previously (Xu et al., 2006 (link), 2007 (link)). The temporal-spatial intensity changes of AdcC-YFP or AdcC-mCherry in cells were directly imaged using a Zeiss 710 or 780 LSM confocal microscope (Zeiss, Jena, Germany) using an EC plan-Neofluar objective 40× oil lens. To quantify the plasma membrane recruitment of YFP- or mCherry-tagged AdcC, intensities were measured using the Zeiss ZEN software package. For each cell, a region of interest (ROI) was drawn at the plasma membrane to measure the fluorescence intensity change over time. Background intensity was measured in each frame and was subtracted from the mean ROI measurements from that frame. The subtracted fluorescence intensities were normalized to the first frame with the appearance of cAMP stimulation, which is defined as 1.
3
Visualizing Organelle Localization in Cancer Cells
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Cancer cells were seeded in a four-well chamber (Thermo Scientific, Nunc, Waltham, MA, USA) slides with 1 × 105 cells in each well. The cells were fixed in 2% paraformaldehyde for 15 min, permeabilised for 5 min with Triton-X (0.2%) in PBS solution. After subsequent PBS washes the slides were incubated in PKD1 (1:500), MTA1 (1:1000), GM130 (1:250), p230 (1:250) and Ubiquitin (1:500) antibodies for 1 h at room temperature. After washing with PBS, the slides were incubated with anti-mouse CY3 and anti-rabbit Alexa Fluor 488 (1:200) (Thermo Fisher Scientific, Carlsbad, CA, USA)-labelled secondary antibody, mounted with Vectashield Mounting Medium containing DAPI and then processed for confocal microscopy laser with Zeiss 710 confocal microscope (Zeiss, Oberkochen, Germany). Similarly, cells were fixed, permeabilised and stained lysotracker Red DND-99 (Thermo Fisher Scientific, Carlsbad, CA, USA).
4
Immunocytochemical Analysis of pMLC2 and F-actin
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For immunocytochemistry, 30,000 cells were seeded in a four-well chamber (Thermo Fisher Scientific) and treated with glucose as described above. After fixation with 4% paraformaldehyde for 15 min at room temperature, cells were blocked with blocking buffer (Cell Signaling Technology) for 60 min at room temperature. Then cells were incubated with p-MLC2 antibody (Cell Signaling Technology, 3671) diluted 1:50 overnight at 4°C. Secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, R37116) was used for visualization. For costaining with filamentous actin (F-actin), cells were stained with Acti-stain 555 phalloidin (Cytoskeleton, PHDH1) as described in the manufacturer’s instructions. Then DAPI (Invitrogen, R73606) was added for nucleus staining. Confocal microscopy images were acquired using LSM900 scanning microscope (Carl Zeiss) equipped with 63×, 40×, and 20× magnification objectives. To determine subcellular localization of pMLC2 and F-actin, confocal microscopy images were analyzed with line profiles using ZEN 3.4 software (Carl Zeiss). The colocalization of pMLC2 and F-actin signals was evaluated with Pearson’s correlation coefficient (r).
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