For determination of proliferative function, DFAT cells and PDLSCs were seeded at cell density of 1 × 104 cells into 35-mm dish (Falcon). The numbers of cells were counted in triplicate every 2 days for 2 weeks. PDT was calculated by PDT software [40 ].
35 mm dish
Manufactured by BD
Sourced in United States
The 35 mm dish is a circular cell culture vessel with a diameter of 35 millimeters. It is designed for in vitro cell culture and experimentation purposes.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 multiphoton microscope, by Leica (1 mentions)
Las x, by Leica (1 mentions)
Nusieve gtg agarose, by Lonza (1 mentions)
35 mm polystyrene dishes, by BD (1 mentions)
Hek293 cell, by Thermo Fisher Scientific (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
P100 dish, by BD (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
Mouse monoclonal vimentin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Dpbs ca2 mg2, by Thermo Fisher Scientific (1 mentions)
6 protocols using 35 mm dish
1
Cell Proliferation Determination Protocol
2
Agarose-embedded Tissue Imaging
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Post fixation, explants were thoroughly washed in PBS 3X times, 1 h each, followed by, embedding in NuSieve GTG agarose (2%, Lonza, cat.no. 859081) in a 35 mm dish (Falcon, cat.no. 351008). Imaging was performed under a Leica SP8 Multiphoton microscope (Leica, Germany). Tiles were merged with a LAS X (v4.8, Leica) with smooth overlap blending, and data was finally visualized with Imaris image analysis software (v9.1.0 and v9.2, Bitplane, UK) using contrast and brightness adjustments.
3
Transient Transfection of TRPM8 in HEK-293 Cells
4
Transient Transfection of TRPM4 Variants in HEK293 Cells
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Human embryonic kidney (HEK293) cells were cultured with Dulbecco's modified Eagle's culture medium supplemented with 4 mM Glutamine, 10% FBS and a cocktail of streptomycin-penicillin antibiotics. For the electrophysiological studies, the cells were transiently transfected with 240 ng of HA-TRPM4 WT or HA-TRPM4 p.A101T, HA-TRPM4 p.Q854R, HA-TRPM4 p.S1044C, HA-TRPM4 p.P1204L, and double variant HA-TRPM4 p.A101T/P1204L in a 35 mm dish (BD Falcon, Durham, North Carolina, USA) mixed with 4 μL of JetPEI (Polyplus transfection, Illkirch, France) and 46 μL of 150 mM NaCl. The cells were incubated for 24 h at 37°C with 5% CO2. All transfections included 100 ng of eGFP as a reporter gene. Expression of GFP was used to identify transfected cells, for patch clamp experiments.
For the biochemical experiments, HEK293 cells were transiently transfected with 240 ng of either HA-TRPM4 WT, or HA-TRPM4 p.A101T, HA-TRPM4 p.Q854R, HA-TRPM4 p.S1044C, HA-TRPM4 p.P1204L, and double variant HA-TRPM4 p.A101T/P1204L, or empty vector (pcDNA4TO) in a P100 dish (BD Falcon, Durham, North Carolina, USA) mixed with 30 μL of JetPEI (Polyplus transfection, Illkirch, France) and 250 μL of 150 mM NaCl. The cells were incubated for 48 h at 37°C with 5% CO2.
For the biochemical experiments, HEK293 cells were transiently transfected with 240 ng of either HA-TRPM4 WT, or HA-TRPM4 p.A101T, HA-TRPM4 p.Q854R, HA-TRPM4 p.S1044C, HA-TRPM4 p.P1204L, and double variant HA-TRPM4 p.A101T/P1204L, or empty vector (pcDNA4TO) in a P100 dish (BD Falcon, Durham, North Carolina, USA) mixed with 30 μL of JetPEI (Polyplus transfection, Illkirch, France) and 250 μL of 150 mM NaCl. The cells were incubated for 48 h at 37°C with 5% CO2.
5
Immunofluorescence Analysis of Vimentin in MSCs
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Immunofluorescence studies were performed for the detection of intracellular Vimentin protein in the MSC samples. Trypsinised cells were seeded on coverslips kept in a 35 mm dish (BD Biosciences, USA), and MSC expansion media were added and incubated at 37°C, 5% CO2. Once confluency reached within 40–50%, the existing media from the dish were removed and cells were washed with PBS. Cells were fixed with 1 ml of 4% paraformaldehyde for 20 mins at 4°C. Permeabilization and blocking was done with 0.05% Tween-20 followed by 2% BSA in PBS for 30 mins at room temperature. MSCs were incubated with the Mouse monoclonal vimentin (Abcam, Cambridge, USA) primary antibody (1/100) for overnight at 4°C. Next day, after washing with PBS, cells were incubated with the secondary fluorescent antibody (1/500) for 40 mins at room temperature (RT). To visualise nuclei, slides were stained with diluted 1/4000 DAPI (Life Technologies, USA) for 3 mins at RT followed by thorough washing of the cells with PBS. The acquisition and imaging of the cells were performed using Floid Microscopy (Life Technologies).
6
Colony Forming Unit Assay for Stem Cells
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The colony forming unit (CFU) assay was determined by re-plating 100 cells in a 35-mm dish (BD Bioscience), followed by 14 days of culturing at 37°C with 5% CO2 . Then, the cells were rinsed from growth media twice using DPBS -Ca2+, -Mg2+ (Invitrogen) and fixed with 100% methanol (Mallindkrodt, Hazelwood, USA, http://pharmaceuticals.covidien.com ) for 20 minutes at room temperature, followed by 3% crystal violet (Sigma Aldrich) staining. Next, the blue stain was rinsed out using tap water for four times until the dishes became colourless. The dishes were then inverted downwards on a clean cloth, and allowed to air-dry for several minutes. Stained colonies with sizes larger than 2 mm were counted. The CFU of ACSs was then calculated using the formula;
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