P12g 1.5 14 f

Manufactured by MatTek

The P12G-1.5–14-F is a laboratory equipment product manufactured by MatTek. It is a multi-well plate with 12 wells, each measuring 1.5 cm in diameter and 14 mm in depth. The product is made of a durable, high-quality material suitable for various research and experimental applications.

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Lab products found in correlation

Axio observer z1, by Zeiss (3 mentions) Alexa fluor 594, by Thermo Fisher Scientific (2 mentions) A1r tie live cell imaging confocal system, by Nikon (1 mentions) Penicillin streptomycin, by Lonza (1 mentions)

5 protocols using p12g 1.5 14 f

NR744 Subcellular Localization Imaging

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Cells were grown to ≈ 80 % confluency on glass-bottomed, 12-well plates (MatTek, P12G-1.5–14-F). After the culture medium had been aspirated, the cells were washed three times with prewarmed Dulbecco’s phosphate-buffered saline (DPBS; Life Tech, 14040117) and incubated with the indicated concentration of NR₇₄₄, along with the indicated subcellular organelle tracker, for 30 min in culture medium. Cells were then washed with DPBS three times and imaged in DPBS. Confocal microscopy was performed with a Nikon A1R-TiE live-cell imaging confocal system. Laser lines used include blue (405 nm), green (488 nm), and far-red (640 nm). ImageJ software was used for data analysis.

Zhou X., Fang Y., Lesiak L, & Stains C.I. (2019). A Phosphinate-Containing Fluorophore Capable of Selectively Inducing Apoptosis in Cancer Cells. Chembiochem : a European journal of chemical biology, 20(13), 1712-1716.

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Immunofluorescence Staining of 53BP1 and DNA Damage

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Seventy percent confluent cell cultures prepared in 12-well plates (Mattek, catalog no. P12G-1.5–14-F) were washed 2×4°C 1x PBS (5 min/wash) and then fixed with 4% paraformaldehyde in 1x PBS for 15 minutes at room temperature. Post-fixation cells were washed 2x with ice-cold 4°C 1x PBS, permeabilized using 0.5% Triton X-100 in 1x PBS for 15 minutes at room temperature, and then incubated with 5% normal goat serum, 0.2% Triton X-100 in 1x PBS for 1 hour at room temperature to block nonspecific antibody sites. Cells were incubated with primary antibodies (1:1,000 53BP1, Cell Signaling Technology, catalog no. 4937; 1:1,000 53BP1^Ser25, Sigma, catalog no. PLA0126) overnight at 4°C followed by washing 3x with 4°C 1x PBS before incubation with the secondary antibody (anti-rabbit Alexa Fluor 594, Thermo Fisher/Invitrogen, catalog no. 11012) for 1 hour at room temperature. Following secondary antibody incubation, cells were washed 3x with PBS as described above, stained with 10 μmol/L Hoescht 33342 in 1x PBS before mounting. Image acquisition and analysis was performed as described above.

Lundine D., Annor G.K., Chavez V., Maimos S., Syed Z., Jiang S., Ellison V, & Bargonetti J. (2022). The C-terminus of Gain-of-Function Mutant p53 R273H Is Required for Association with PARP1 and Poly-ADP-Ribose. Molecular Cancer Research, 20(12), 1799-1810.

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Immunofluorescence Staining for 53BP1

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70% confluent cell cultures prepared in 12 well plates (Mattek, Cat# P12G-1.5–14-F) were washed 2× 4°C 1x PBS (5 min/wash) and then fixed with 4% Paraformaldehyde in 1x PBS for 15 min at room temperature. Post-fixation cells were washed 2x with ice-cold 4°C 1x PBS, permeabilized using 0.5% Triton X-100 in 1x PBS for 15 min at room temperature, and then incubated with 5% Normal Goat Serum, 0.2% Triton X-100 in 1x PBS for 1 hour at room temperature to block non-specific antibody sites. Cells were incubated with primary antibodies (1:1000 53BP1, Cell Signaling Cat #4937; 1:1000 53BP1^Ser25, Sigma Cat# PLA0126) overnight at 4°C followed by washing 3x with 4°C 1x PBS before incubation with the secondary antibody (anti-rabbit Alexa Fluor 594, Thermo Fisher/Invitrogen Cat #11012) for 1 hr at room temperature. Following secondary antibody incubation, cells were washed 3x with PBS as described above, stained with 10μM Hoescht 33342 in 1x PBS before mounting. Image acquisition and analysis was performed as described above.

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Automated Cell Division Detection Protocol

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HaCaT mCherry-Histone H2B cells were seeded in 12-well glass bottom multiwell plates from MatTek (P12G-1.5-14-F; MatTek Corporation) at 600,000 cells per well. Notably, all cells were seeded 12 to 20 h before the start of serum depletion. Cells were subjected to different time periods of starvation, followed by serum restimulation and widefield microscopy. Image acquisition was carried out on a Zeiss AxioObserver.Z1 microscope using a 10× 0.5 NA FLUAR air objective, a filter set for the detection of mCherry fluorescence, and a CO₂ incubation chamber. Four randomly selected sites per well were acquired for a total time period of 50 h, using a time interval of 16 min between frames. First, mitotic cells in a set of images were annotated and used as input data for development of a deep learning model using the StarDist (2D) network, available in the CoLab notebook (64 ). Second, the deep learning model was further used to detect cell division frequencies in each frame of complete datasets, by running the model in the StarDist plugin of Fiji ImageJ (65 ).

Lång E., Pedersen C., Lång A., Blicher P., Klungland A., Carlson A, & Bøe S.O. (2022). Mechanical coupling of supracellular stress amplification and tissue fluidization during exit from quiescence. Proceedings of the National Academy of Sciences of the United States of America, 119(32), e2201328119.

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Quiescence-dependent Collective Migration Induction

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The experimental approach used to induce quiescence-dependent activation of collective migration has been previously described (23 (link), 24 (link)). Immortalized human keratinocytes (HaCaT) (38 (link)) or HaCaT cells expressing mCherry-tagged histone H2B (24 (link)) were cultured in Iscove’s modified Dulbecco’s medium (MedProbe) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and penicillin/streptomycin (90 U/ml; Lonza). For each experiment, 96-well plates (Greiner Sensoplate; M4187-16EA, Merck) or 12-well plates (P12G-1.5-14-F; MatTek Corporation) were coated with collagen IV (20 μg/ml; C7521, Merck) before seeding the cells at 75,000 or 600,000 cells per well, respectively. The cells were then cultured in normal growth medium at 37°C and 5% CO₂ overnight. To induce quiescence, the cells were serum deprived for 72 hours before being activated with normal growth medium containing 15% FBS. For experiments involving the manipulation of calcium concentration, the serum-deprived cells were stimulated with CnT-Prime Epithelial Proliferation Medium (CELLnTEC), a low calcium medium, human recombinant EGF (10 ng/ml; 236-EG, R&D Systems), and the indicated concentrations of calcium.

Lång E., Lång A., Blicher P., Rognes T., Dommersnes P.G, & Bøe S.O. (2024). Topology-guided polar ordering of collective cell migration. Science Advances, 10(16), eadk4825.

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