Akt Inhibitor VIII, Gö6983, and U0126 was purchased from Calbiochem (Darmstadt Germany), Tocris Bioscience (Bristol, United Kingdom), and Cell Signaling (Danvers MA), respectively. Cyclosporine A and Actinomycin D were purchased from Sigma (St. Louis MO). For flow cytometry, cells were prepared, stained and analyzed as previously described (14 (link)). Antibodies for NK cell stimulations and phenotyping were purchased from BD Pharmingen (San Diego CA), BioLegend (San Diego CA), eBioscience (San Diego CA), Bio X Cell (West Lebanon NH), and Molecular Probes, Invitrogen (Carlsbad CA). Data were analyzed with FlowJo software (Tree Star, Ashland OR) and Prism (Graphpad Software, La Jolla CA). Rat anti-alpha tubulin (clone YL1/2, Bio-Rad, Hercules CA) and 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen) were used for confocal microscopy.
7 amino 4 chloromethylcoumarin cmac
Manufactured by Thermo Fisher Scientific
Sourced in United States
7-amino-4-chloromethylcoumarin (CMAC) is a fluorescent dye compound used in various biochemical and cell biology applications. It serves as a labeling agent, allowing the visualization and detection of target molecules or cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Softworx software, by Cytiva (2 mentions)
Fm4 64, by Thermo Fisher Scientific (2 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Deltavision elite, by Olympus (1 mentions)
Deltavision elite imaging system, by Olympus (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
Xlumplanfi 20 objective, by Olympus (1 mentions)
Fluorescein isothiocyanate dextran fitc dextran, by Merck Group (1 mentions)
Rabbit anti gfp polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Mowiol, by Merck Group (1 mentions)
Mouse anti cd71, by BD (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
8 protocols using 7 amino 4 chloromethylcoumarin cmac
1
Comprehensive NK Cell Phenotyping and Inhibitor Assessment
2
Visualizing Fungal Structures and Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To stain the septa, mycelia or conidia were incubated in 10 μg/mL Calcofluor white (Sigma) solution for 5 min at RT followed by 3 thorough washes with sterile water. Samples were visualized and photographed as previously described [42 (link)]. To observe the fungal infection process, conidial suspension with a concentration of 2 × 105 conidia/mL was dropped onto the lower epidermis of barley leaves and then incubated in a moist, dark chamber at 28 °C. Microscopy observations were performed at 12, 24, 36, and 48 hpi [44 (link)]. For autophagy assays, strains expressing GFP-MoAtg8 were cultured in liquid complete medium for 48 h, and then shifted to nitrogen starvation cultured for 4 h to induce autophagy. Mycelia were harvested from liquid complete medium and nitrogen starvation medium. 7-amino-4-chloromethylcoumarin (CMAC, Invitrogen) was used to stain vacuolar lumens as previously described [45 (link)]. Epifluorescence microscopic examinations were performed by using a Nikon Ni90 fluorescence microscope (Nikon, Tokyo, Japan).
3
Visualizing Yeast Vacuolar Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast cells were grown to exponential phase in yeast extract peptone dextrose (YPD) or synthetic complete medium supplemented with all amino acids (SDC). To stain the vacuolar membrane of the cells, 30 µM FM4-64 (Molecular Probes, Eugene, OR) was added and incubated for 30 min. The cells were washed twice with fresh medium and incubated in medium without dye for 1 h (Vida and Emr, 1995 (link)). For luminal staining of vacuoles, cells were incubated with 0.1 mM 7-amino-4-chloromethylcoumarin [CMAC] (Invitrogen) for 10 min, followed by washing with SDC medium.
Cells were imaged live in SDC media in a DeltaVision Elite imaging system based on an Olympus IX-71 inverted microscope equipped with a 100× N.A. 1.49 objective, a sCMOS camera (PCO, Kelheim, Germany), an InsightSSI illumination system, 40,6-diamidino-2-phenylindole, GFP, mCherry, and Cy5 filters, and SOftWoRx software (Applied Precision, Issaquah, WA). z-stacks images with 0.2-µm spacing were deconvolved (SOftWoRx). All images were processed and quantified with ImageJ software (NIH, Bethesda, MD). One representative plane of the z-stacks from deconvolved image is shown in each figure.
Cells were imaged live in SDC media in a DeltaVision Elite imaging system based on an Olympus IX-71 inverted microscope equipped with a 100× N.A. 1.49 objective, a sCMOS camera (PCO, Kelheim, Germany), an InsightSSI illumination system, 40,6-diamidino-2-phenylindole, GFP, mCherry, and Cy5 filters, and SOftWoRx software (Applied Precision, Issaquah, WA). z-stacks images with 0.2-µm spacing were deconvolved (SOftWoRx). All images were processed and quantified with ImageJ software (NIH, Bethesda, MD). One representative plane of the z-stacks from deconvolved image is shown in each figure.
4
Vacuole Staining and Imaging in Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were grown to exponential phase in yeast extract peptone dextrose (YPD) or synthetic complete medium supplemented with all amino acids (SDC) [0.675% (w/v) yeast nitrogen base without amino acids, 2.0% (w/v) glucose, 0.075% (w/v) CSM (MPBiomedicals)]. To induce starvation, yeast cells were transferred to synthetic minimal medium lacking nitrogen and containing 2% glucose (SD-N) for 3 h. Vacuoles were stained with 30 µM FM4-64 (Molecular Probes Inc., Eugene, OR) for 30 min. Cells were washed twice with fresh medium and incubated in medium without dye for 1 h (Vida and Emr, 1995) (link). To stain the vacuole lumen, cells were incubated with 0.1 mM 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen) for 15 min, followed by washing with SDC medium.
Cells were imaged live in SDC or SD-N media on a DeltaVision Elite imaging system based on an Olympus IX-71 inverted microscope equipped with a 100× N.A. 1.49 objective; an sCMOS camera (PCO, Kelheim, Germany); an InsightSSI illumination system; 4′,6-diamidino-2phenylindole, GFP, mCherry and Cy5 filters; and SOftWoRx software (Applied Precision, Issaquah, WA). Single plane or z-stack images with 0.2 µm spacing were deconvolved (SOftWoRx). All images were processed and quantified with ImageJ software (NIH, Bethesda, MD). One representative plane of the z-stack from a deconvolved image is shown in each figure.
Cells were imaged live in SDC or SD-N media on a DeltaVision Elite imaging system based on an Olympus IX-71 inverted microscope equipped with a 100× N.A. 1.49 objective; an sCMOS camera (PCO, Kelheim, Germany); an InsightSSI illumination system; 4′,6-diamidino-2phenylindole, GFP, mCherry and Cy5 filters; and SOftWoRx software (Applied Precision, Issaquah, WA). Single plane or z-stack images with 0.2 µm spacing were deconvolved (SOftWoRx). All images were processed and quantified with ImageJ software (NIH, Bethesda, MD). One representative plane of the z-stack from a deconvolved image is shown in each figure.
5
Cytokine and Adhesion Molecule Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the assessment of cytokine production, in vitro differentiated Th1 and Th17 cells were stimulated for 12 h with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin, and 10 μg/ml brefeldin A. The cells were then fixed and permeabilized using the Biolegend Fixation buffer/Permeabilization wash buffer kit and labeled with phycoerythrin (PE)-conjugated rat anti-mouse IFNγ (clone REA638, Miltenyi Biotech) and eFluor660-conjugated rat anti-mouse IL-17A (clone eBio17B7, eBioscience). Control cells were stimulated under the same conditions, but without brefeldin A. For the analysis of adhesion molecule expression, in vitro differentiated and ex vivo labeled with 7-amino-4-chloromethylcoumarin (CMAC; Molecular Probes) Th1 and Th17 cells were stained with the following rat anti-mouse monoclonal antibodies: APCFire750-conjugated anti-CD49d, FITC-conjugated anti-CD11a/CD18, all from Biolegend. For ex vivo studies, cell viability was confirmed using 7-aminoactinomycin D (7AAD; eBioscience) and the mean fluorescence intensity (MFI) of adhesion molecule expression was calculated relative to CMAC+ infiltrating cells. We used a MACSQuant Analyzer (Miltenyi Biotec) for the acquisition of flow cytometry data, followed by analysis using FlowJo software.
6
In Vivo Imaging of T Cell Infiltration in EAE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Th1 or Th17 cells were labeled for 45 min at 37°C with 7-amino-4-chloromethylcoumarin (CMAC) (Molecular Probes). Then, 2 × 107 Th1 or Th17 cells were intravenously injected into MOG35-55-immunized EAE recipient mice at the onset of the disease. After 48 h, when mice reached the EAE peak, 70 KDa fluorescein isothiocyanate dextran (FITC–dextran) (Sigma Chemical Co., St. Louis, MI, USA) was intravenously injected immediately before imaging of blood vessels. A mode-locked Ti : Sapphire Chameleon Ultra II laser (Coherent Inc.) was tuned to 750–800 nm and an Olympus XLUMPlanFI 20× objective (water immersed, numerical aperture, 0.95) was used for TPLSM imaging. To visualize infiltrating T cells in the dorsal spinal SAS, time-lapse sequences were performed at 70 μm stack height (2.5 μm z step) at 35-40 s intervals as previously described (17 (link)). Immediately after the first acquisition, 200 μl of artificial CSF containing 100 μg/ml of blocking anti-α4 integrins (clone PS/2, rat anti-mouse) or blocking anti-α4β7 integrin (clone DATK32, rat anti-mouse) were locally applied to the exposed spinal cord and incubated for 30 min before a second round of imaging, to allow adequate local meningeal diffusion (17 (link), 23 (link)–26 (link)).
7
Culturing Jurkat and Raji Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Jurkat CD4 T cell line (JK) and the Raji B cell line (American Type Culture Collection) were grown at 37°C and 5% CO2 in RPMI 1640 medium (Lonza Group, Switzerland), supplemented with 10% inactivated fetal calf serum (FCS) (Gibco, Gaithersburg, MD, United States), 10 mM glutamine, 100 U/mL penicillin and 100 μg/ml streptomycin (all from Lonza Group). Mouse anti-CD3 monoclonal antibody was provided by Dr. Francisco Sánchez-Madrid (Hospital Universitario de la Princesa, Madrid, Spain); mouse anti-PRL-1/2 (clone 42) monoclonal antibody was purchased from Millipore (Burlington, MA, United States); mouse anti-CD71 was from BD Bioscience (San Jose, CA, United States) and rabbit anti-GFP polyclonal antibody was purchased from Life Technologies (Carlsbad, CA, United States). Secondary antibody goat anti-mouse-Ig Alexa Fluor 594 (highly cross-absorbed) and the fluorescent tracker 7-amino-4-chloromethyl coumarin (CMAC) were obtained from Molecular probes (Eugene, OR, United States). Poly-L-lysine, Mowiol and brefeldin A (BFA) were obtained from Sigma Aldrich (St. Louis, MO, United States) and Staphylococcus Enterotoxin E (SEE) from Toxin Technologies (Sarasota, FL, United States).
8
ER-Phagy Induction and Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ER-phagy was induced as previously described [14 (link)]. Briefly, cells were grown to exponential phase in YPD. Rapamycin was added to a final concentration of 200 ng/mL and the cultures were incubated for 16–18 h at 30 °C. The location of the vacuole was identified either by staining with 7-amino-4-chloromethylcoumarin (CMAC) (Molecular Probes, Eugene, OR, USA) or by differential interference contrast microscopy. ER-phagy was detected by the presence of Rtn1-GFP fluorescence in the vacuolar lumen by fluorescence microscopy.
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!