mCCDcl1 cells were seeded on to Corning Costar™ Transwell™ Permeable Supports (0.4 μm pore size) in 24 well plates at 1×105 cells/cm2 in complete growth medium. After 7–9 days, cell monolayers were washed in PBS for two times and fixed in 100% methanol (chilled at -20°C) for 5 min at RT, followed by three times wash in ice cold PBS for 5 min each as done before [27 (link), 34 (link)]. The cells were then blocked in blocking buffer (PBS, 10% goat serum and 0.05% Triton X-100) for one hour. Whereupon primary antibodies were add to the blocking buffer at 1:100 dilution and incubated overnight at 4°C. The following primary antibodies were used: anti-SK1 (Alomone, APC-039), anti-SK3-ATTO-594 (Alomone, APC-025-AR), anti-IK1 (Alomone, ALM-051), anti-BKα (Alomone, APC-021) and anti-TRPV4 (Alomone, ACC-034). After primary antibody incubation, the cells were washed 3 times in PBS, 5 min each, followed by incubation with secondary antibody (Life Technologies, alexa fluor 488 or 594 labeled goat anti-rabbit) in blocking buffer. The cells were then washed another three times in PBS, 5 min each, and mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). Cells were imaged at 100x using a Nikon A1R Confocal Laser Microscope or a Zeiss Axioskop 40 microscope equiped with a AxioCam MRm CCD camera.
Alm 051
Manufactured by Alomone
Sourced in Israel
ALM-051 is a laboratory equipment product. It is designed for scientific research and analysis purposes. The core function of ALM-051 is to facilitate the measurement and analysis of various parameters within a controlled laboratory environment. No further details are provided about the intended use or specific applications of this product.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam mrm ccd camera, by Zeiss (1 mentions)
Transwell permeable support, by Corning (1 mentions)
Axioskop 40 microscope, by Zeiss (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Apc 039, by Alomone (1 mentions)
Apc 021, by Alomone (1 mentions)
Acc 034, by Alomone (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Zymo Research (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Apr 010, by Alomone (1 mentions)
Apr006, by Alomone (1 mentions)
4 protocols using alm 051
1
Immunofluorescence Staining of mCCDcl1 Cells
2
Western Blot Analysis of KCa3.1 in SKOV-3 Cells
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Cultured SKOV-3 cells were scraped in Laemmli buffer and boiled for 5 min. For electrophoresis, samples were fractionated in a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (BioRad; Hercules, CA, USA). Membranes were blocked for 1 h at room temperature in 150 mM NaCl, 20 mM Tris, pH 7.4, and 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated overnight at 4 °C with the appropriate mouse monoclonal antibody (1:1000) directed against the KCa3.1 channel protein (ALM-051, Alomone; Jerusalen, Israel). After washing with TBS-T, membranes were incubated for 1 h at 37 °C with HRP-conjugated goat anti-rabbit antibody (Zymed; Grand Island, NY, USA) in TBS-T. The immunoreactive proteins were detected by chemiluminescence, and images were analyzed with ImageJ Software.
3
Immunostaining of P2Y2, P2Y4, and KCa3.1 Channels
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For P2Y2, P2Y4 or KCa3.1 channel immunostaining, cells were treated with the corresponding antibody (anti-P2Y2 (1/100) APR010; anti-P2Y4 (1/100) APR006; or anti-KCa3.1 (1:1000) ALM051; all from Alomone, Jerusalem, Israel). In all cases the cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. The fixed cultures were permeabilized with 0.1% Tween-20, blocked with 5% goat serum in PBS for 30 min and incubated overnight at 4 °C with the antibodies diluted in PBS containing 5% goat serum and 0.1% Tween-20. Then, cells were rinsed and incubated for 2 h at room temperature with 1:100 anti-mouse IgM-G conjugated with Alexa 488 (Molecular Probes). After three washes with PBS, samples were stained with 4′,6-diamidino-2-phenyl-indole dihydrochloride (DAPI 14 mM, from Molecular Probes). Finally, the samples were mounted on VectaShield (Vector Laboratories; Burlingame, CA, USA), and the preparations were visualized under a laser scanning confocal microscope LSM510 (Zeiss; Oberkochen, Germany).
4
Evaluating IK Ca Expression on Cell Surface
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The IK Ca protein expression on cell surface was determined by flow cytometry as previously described (Jia et al., 2013) (link). Briefly, cells were seeded on 6-well plates covered with defined PDMS substrates at a density of 8×10 4 cells/well and cultured for 2 days. Cells were harvested and fixed with 4% paraformaldehyde for 30 min. Following three washes with PBS, cells were incubated with a primary mouse antibody specifically against the IK Ca extracellular domain (ALM051, 1:100, Alomone Labs, Israel) for 40 min. Cells were incubated with the irrelevant mouse isotope antibody (1:100, Alomone Labs, Israel) instead of specific antibody as negative control (NC). After three washes with PBS, cells were incubated with a secondary fluorescein isothiocyanate-conjugated anti-mouse IgG antibody Transfection with siRNA IK Ca -specific siRNA (siIK Ca ) (sense: 5'-GGAGGUCCAGCUGUUCAUGtt-3', antisense:
5'-CAUGAACAGCUGGACCUCCtt-3') and a scrambled control siRNA (ctrl siCTL) (sense:
5'-CAUUCACUCAGGUCAUCAGtt-3', antisense: 5'-CUGAUGACCUGAGUGAAUGtt-3') were synthesized by GenePharma (Shanghai, China). Cells were seeded on 6-well plates at a density of 8×10 4 cells/well and transfected with 25 nM of siIK Ca or siCTL using siPORTt Amine (Ambion)
according to the manufacturer's protocols. Cells 24 hr post transfection were seeded on PDMS substrates for further experiments.
5'-CAUGAACAGCUGGACCUCCtt-3') and a scrambled control siRNA (ctrl siCTL) (sense:
5'-CAUUCACUCAGGUCAUCAGtt-3', antisense: 5'-CUGAUGACCUGAGUGAAUGtt-3') were synthesized by GenePharma (Shanghai, China). Cells were seeded on 6-well plates at a density of 8×10 4 cells/well and transfected with 25 nM of siIK Ca or siCTL using siPORTt Amine (Ambion)
according to the manufacturer's protocols. Cells 24 hr post transfection were seeded on PDMS substrates for further experiments.
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