Millicell cm culture plate insert

Manufactured by Merck Group

Sourced in United States, Switzerland

Millicell-CM culture plate inserts are a laboratory equipment product designed for cell culture applications. They provide a porous membrane support for growing cells in a well plate format. The inserts allow for the separation of cell layers and the exchange of media and nutrients across the membrane.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Gey s balanced salt solution, by Merck Group (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Rabbit polyclonal antibody against cleaved caspase 3, by Cell Signaling Technology (1 mentions) Nis element software, by Nikon (1 mentions) Fv10i confocal microscope, by Olympus (1 mentions) Immpact dab, by Vector Laboratories (1 mentions) Millicell cm, by Merck Group (1 mentions) Glutamine, by Thermo Fisher Scientific (1 mentions) Hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions) Kynurenic acid, by Merck Group (1 mentions) Bx51 microscope, by Olympus (1 mentions) Hanks balanced salt solution (hbss), by Merck Group (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) S6d stereozoom, by Leica (1 mentions) 24 well culture plate, by Corning (1 mentions) Matrigel, by BD (1 mentions) Su5402, by Fujifilm (1 mentions) Dmem medium, by Thermo Fisher Scientific (1 mentions)

9 protocols using millicell cm culture plate insert

In Vitro Glioblastoma Tumor Modeling in Mouse Brain Slices

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Fifty thousand GICs were orthotopically implanted into the forebrain of wild-type mice, and at 7 days post-implantation, brain slice explants were established as previously described^{33 (link)}. Coronal slices (200 µm) were cultured on Millicell-CM culture plate inserts (Millipore) and treated with vehicle or a131 for 4 days. Images were acquired on an FV10i Olympus confocal microscope (Olympus) and tumor area was quantified by Nikon NIS-element software. Experiments were performed in triplicate. At the end of the experiment (Day 4), slices were fixed overnight in 4% paraformaldehyde, embedded in paraffin and then sectioned at a thickness of 4 µm. Deparaffinized sections were stained with rabbit polyclonal antibody against cleaved caspase-3 (Cell Signaling). Immune complexes were detected using Histofine (Nichirei Biosciences) and ImmPACTDAB (Vector Laboratories). All animal experiments were performed in accordance with the animal care guidelines of Keio University.

Kitagawa M., Liao P.J., Lee K.H., Wong J., Shang S.C., Minami N., Sampetrean O., Saya H., Lingyun D., Prabhu N., Diam G.K., Sobota R., Larsson A., Nordlund P., McCormick F., Ghosh S., Epstein D.M., Dymock B.W, & Lee S.H. (2017). Dual blockade of the lipid kinase PIP4Ks and mitotic pathways leads to cancer-selective lethality. Nature Communications, 8, 2200.

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Culturing Retinal Explants In Vitro

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Explants from E8 retinas were cultured in vitro on 30-mm Millicell CM culture plate inserts (Millipore, Billerica, MA; pore size 0.4 µm) according to the method described by Stoppini et al. (1991) [53] with some modifications [54] (link). Retinas were dissected out into cold Gey's balanced salt solution (Sigma, St. Louis, MO) supplemented with 5 mg/mL glucose (Sigma) and 50 IU-µg/mL penicillin-streptomycin (Invitrogen, Paisley, United Kingdom). After removing the pigment epithelium, square explants (3 mm×3 mm) were isolated from the central area of each retina and then placed on Millicell inserts (Millicell CM, Millipore, Bedford, MA, USA; pore size 0.4 µm), vitreal surface down. Millicell inserts were put in six-well plates containing 1 mL/well culture medium composed of 50% basal medium with Earle's salts, 25% Hank's balanced salt solution, 25% horse serum, 1 mM L-glutamine, 10 IU-µg/mL penicillin-streptomycin (all purchased from Invitrogen), and 5 mg/mL glucose. E8 retina explants were then incubated at 37°C in a humidified atmosphere with 5% CO₂ for 1 hour in vitro (hiv) to 24 hiv (E8+1hiv to E8+24hiv) according to the aim of each experiment.

Sierra A., Navascués J., Cuadros M.A., Calvente R., Martín-Oliva D., Ferrer-Martín R.M., Martín-Estebané M., Carrasco M.C, & Marín-Teva J.L. (2014). Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina. PLoS ONE, 9(8), e106048.

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Demyelination of Organotypic Slice Cultures

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Organotypic slice cultures (OSCs) were generated from 10 days old mice as described before [25 (link)]. The cerebellum was cut into 400 μm thick slices using a McIllwain tissue chopper (GaLa Instrumente). OSC were dissociated in ice-cold dissecting medium (Hank’s Balanced Salt Solution (HBSS), Life Technologies) complemented with penicillin/streptomycin (100 U/ml, Life Technologies), 2.5 mg/ml glucose (Sigma Aldrich) and 10 mM kynurenic acid (Sigma Aldrich). OSCs were cultured on Millicell-CM culture plate inserts (Millipore) in culture medium (50% (v/v) MEM, 25% (v/v) HBSS, 25% (v/v) heat-inactivated horse serum, 2 mM glutamine, penicillin/streptomycin (100 U/ml) (all from Life Technologies) and 2.5 mg/ml glucose (Sigma Aldrich) for 3–5 days at 37°C in a humidified atmosphere with 5% CO₂, and then demyelinated with lysolecithin (0.5 mg/ml, 16 h). After incubation the lysolecithin containing medium was removed and replaced with fresh medium. At this point OSCs were used for all experiments. In some experiments OSCs were treated with 100 U/ml mouse rIFNβ as indicated. For the usage of PLP-EGFP slices, fluorescent images were taken on indicated time points with an Olympus BX51 microscope at low magnification and in sterile conditions.

Kocur M., Schneider R., Pulm A.K., Bauer J., Kropp S., Gliem M., Ingwersen J., Goebels N., Alferink J., Prozorovski T., Aktas O, & Scheu S. (2015). IFNβ secreted by microglia mediates clearance of myelin debris in CNS autoimmunity. Acta Neuropathologica Communications, 3, 20.

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Quail Embryo Retina Explant Culture

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Quail embryo retina explants (QEREs) from E8 were cultured in vitro on 30-mm Millicell CM culture plate inserts (Millipore, Bedford, MA, USA; pore size 0.4 mm), according to the method previously described (Stoppini et al., 1991 (link)) with some modifications (Carrasco et al., 2011 (link)). Briefly, retinas were dissected and kept in cold Gey’s balanced salt solution (Sigma, St. Louis, MO) supplemented with 5 mg/mL glucose (Sigma) and 50 IU-mg/mL penicillin-streptomycin (Invitrogen, Paisley, United Kingdom). After removing the retinal pigmented epithelium, square explants (3 mm x 3 mm) containing the dorsal part of base of the pecten and the optic nerve head area were isolated from the central region of each retina and then placed on Millicell inserts, with the vitreal surface downward. Subsequently, these Millicell inserts were put in six-well plates containing 1 mL/well culture medium composed of 50% basal medium with Earle’s salts, 25% Hank’s balanced salt solution, 25% horse serum, 1 mM L-glutamine, 10 IU-mg/mL penicillin-streptomycin (all purchased from Invitrogen), and 5 mg/mL glucose. E8 QEREs were then incubated at 37°C in a humidified atmosphere with 5% CO₂ for 1 h in vitro (hiv) to 24 hiv (E8 + 1hiv to E8 + 24hiv, respectively).

Sierra-Martín A., Navascués J., Neubrand V.E., Sepúlveda M.R., Martín-Oliva D., Cuadros M.A, & Marín-Teva J.L. (2023). LPS-stimulated microglial cells promote ganglion cell death in organotypic cultures of quail embryo retina. Frontiers in Cellular Neuroscience, 17, 1120400.

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Organotypic Cerebellar Slice Culture Protocol

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Organotypic cerebellar slice-cultures were prepared from postnatal day (P) 8–10 mice as described previously [21 (link)]. The mice were euthanized by CO₂ inhalation and then immediately perfused with saline (0.9% NaCl). Next the cerebellum was dissected out and sagittally sliced at 250 μm using a McIlwain tissue chopper. Lobule X was isolated from residual meninges in cold 1X Hank’s balanced salt solution (HBSS, Life Technologies) and then placed on the membrane surface of Millicell-CM culture plate inserts (Millipore) equilibrated in plating medium (Basal medium eagle containing 25% horse serum, 1/4X HBSS, 1mM L-glutamine, 0.1% D-glucose, 1% penicillin/streptomycin, and 0.05% fungizone). At 7 days in vitro (DIV), the medium was replaced with maintaining medium (Basal medium eagle containing 15% horse serum, 1/4X HBSS, 1mM L-glutamine, 0.1% D-glucose, 1% Penicillin/streptomycin, and 0.05% fungizone). The medium was changed every 2–3 days and cultures were grown for 22– 24 DIV at 37°C in a humidified atmosphere with 5%CO₂/95%O₂. The slices were then fixed for 30 min at room temperature with 4% paraformaldehyde in 0.12 M PB after which they were kept in PBS at 4°C.

Lee S.K., Sillitoe R.V., Silva C., Martina M, & Sekerkova G. (2015). α-synuclein expression in the mouse cerebellum is restricted to VGluT1 excitatory terminals and is enriched in unipolar brush cells. Cerebellum (London, England), 14(5), 516-527.

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Hippocampal Slice Culture Preparation

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All animal work was carried out in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) and under the project and personal licenses approved by the Home Office (UK). Slice cultures of the hippocampus were prepared from male Wistar rats (P6-8). The hippocampi were isolated in ice-cold Earle’s balanced salt solution (EBSS) with added: HEPES (21 mM), D-(+)-Glucose (27.8 mM) pH adjusted to 7.2 - 7.4 with NaOH and cut into slices of 350 μm thickness with a McIlwain tissue chopper. Slices were placed into Millicell CM culture plate inserts (PTFE filter, pore size 0.4 μm, diam. 12 mm) in a six-well Millicell culture plate (both supplied by Merck Millipore) with 1 mL of culture medium and stored at 34.5°C at 5% CO₂. Culture medium composed of 78.8% minimum essential medium (MEM) with GlutaMAX (Gibco), 20% heat-inactivated horse serum, 1% B27 with added CaCl₂ (1 mM), HEPES (30 mM,) D-(+)-Glucose (26 mM,) NaHCO₃ (5.8 mM) and MgSO₄ (2 mM). Culture media was renewed every 3-4 days.
During experiments, slices (10–14 days in vitro [DIV]) were perfused (1–2 mL/min) with heated (32°C–34°C) artificial cerebrospinal fluid (ACSF) which comprised of NaCl (145 mM), KCl (2.5 mM), KH₂PO₄ (1.2 mM), NaHCO₃ (16.0 mM), glucose (11.0 mM), CaCl₂ (3.0 mM) and MgCl₂(2.0 mM) aerated with 95% O₂, 5% CO₂.

Foster W.J., Taylor H.B., Padamsey Z., Jeans A.F., Galione A, & Emptage N.J. (2018). Hippocampal mGluR1-dependent long-term potentiation requires NAADP-mediated acidic store Ca2+ signaling. Science signaling, 11(558), eaat9093.

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Explant Culture Regulation by CUL3 Silencing

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Explant culture was performed as described previously (Genbacev et al. 1992) (link). In brief, placental anchoring villi (5-8 w) were identified by a phase contrast microscope (Leica S6 D Stereozoom; Leica, Heerbrugg, Switzerland), dissected, and placed on the Millicell-CM culture plate inserts (EMD Millipore Corp.) pre-coated with phenol red-free and growth factor-reduced Matrigel (Becton Dickinson, Bedford, MA, USA). The inserts were then placed into a 24-well culture plate (Costar, Cambridge, MA, USA). The explants were cultured in serum-free DMEM mixed 1:1 with Ham's F-12 (DMEM/F12; HyClone) medium with 500 nM of scrambled control siRNA or CUL3 siRNA (5 0 -AUAACUUGUACAUG-CAACCAAGGUC-3 0 ). Explants were photographed daily for up to 4 days. The distance from the cell column base to the tip of the outgrowth was measured with SPOT Imaging Software (SPOT Imaging Solutions, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). All the explant experiments were repeated three times and replicated in four separate sets of explants.

Zhang Q., Yu S., Huang X., Tan Y., Zhu C., Wang Y.L., Wang H., Lin H.Y., Fu J, & Wang H. (2015). New insights into the function of Cullin 3 in trophoblast invasion and migration. Reproduction (Cambridge, England), 150(2).

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Conjunctival Impression Cytology Protocol

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The patients were also subjected to conjunctival impression cytology, taking four samples per patient, two specimens per eye, at the level of the nasal and temporal conjunctiva. Impression cytology was obtained after the administration of topical anaesthesia with 2 drops of 4% ossibuprocain. The filters used for the collection were special nitrocellulose filters (Millicell-®CM Culture Plate Insert, 0.4 μm, Ø 12 mm, PICM 012 50, Millipore). The operator kept the membrane on the conjunctiva for 5 seconds using the plastic support.
The preparations were then fixed with the Bio-Fix cytological fixator (Kaltek s.r.l.), placed in its original container and numbered for subject, eye, area, and date of exam. The samples were stored in a freezer at −80°C, pending semi-Schiff (PAS) staining.
PAS colouration was performed by applying the following procedure to all cytology samples:

periodic acid at 0.5% for 2 min; washing in distilled water

Schiff reagent for 10 min; washing in running water trays for 5 minutes

Pure haematoxylin for 2 min; washing in running water trays for 5 minutes

The samples were then observed under NIKON Eclipse E600 optical microscope to perform caliciform-forming cell counts. The density of the goblet cells was defined as the average of the cells per field in 3 different random fields.

Nebbioso M., Sacchetti M., Bianchi G., Zicari A.M., Duse M., Del Regno P, & Lambiase A. (2018). Tear Ferning Test and Pathological Effects on Ocular Surface before and after Topical Cyclosporine in Vernal Keratoconjunctivitis Patients. Journal of Ophthalmology, 2018, 1061276.

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Explant culture and drug-bead transplantation

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Explant culture of the mandibular arch and transplantation of drug-soaked bead were performed mostly as previously described^{8 (link)}. Mandibular arch along with flanking tissues were dissected from E11.0 M. domestica, E10.0 mouse, and E3 quail embryos. One AG1-X2 (formate form) anion exchange bead (Bio Rad), soaked with either 10 mM SU5402 (197-16731, WAKO) in DMSO or DMSO alone, was transplanted into each mandibular arch. Explants were placed onto Millicell-CM culture plate insert (0.4um pore size, Millipore), and cultured in DMEM medium (Gibco) for 24 hours. Explants were subsequently fixed in 4% PFA/PBS, and processed for whole-mount in situ hybridization, as indicated above.

Wakamatsu Y., Egawa S., Terashita Y., Kawasaki H., Tamura K, & Suzuki K. (2019). Homeobox code model of heterodont tooth in mammals revised. Scientific Reports, 9, 12865.

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