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Membrane inserts

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Membrane inserts are a versatile lab equipment used in cell culture applications. They facilitate the study of cellular interactions and transport processes by creating a physical barrier between two cell culture compartments. The membrane insert serves as a permeable surface that allows the exchange of nutrients, signaling molecules, and other substances between the separated compartments.

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Lab products found in correlation

Laminin, by Merck Group (2 mentions) Withpoly l orthinine, by Merck Group (2 mentions) Basal medium eagle, by Thermo Fisher Scientific (2 mentions) Vt1200 vibrotome, by Leica camera (2 mentions) Sp8 confocal microscope, by Leica camera (2 mentions) Ham s f12 medium, by Thermo Fisher Scientific (1 mentions) Evos fl color imaging system, by Thermo Fisher Scientific (1 mentions) Matrigel matrix solution, by BD (1 mentions) Imagexpress micro xls widefield high content analysis system, by Molecular Devices (1 mentions) Softmax pro software, by Molecular Devices (1 mentions) Matrigel, by BD (1 mentions) C9301, by Merck Group (1 mentions)

11 protocols using membrane inserts

1

Organotypic Forebrain Slice Culture Protocol

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Organotypic cultures of mouse coronal forebrain slices were prepared
following published methods45 (link)with some modifications. Whole brains from E14–E18 mouse embryos were
embedded in 4% low-melting point agarose and slices were cut at
250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100
ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30
ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1
M NaHCO3). Slices with visible forebrain structures were placed in membrane
inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated with
Poly-L-orthinine and Laminin (Sigma) overnight. They were cultured in a Basal
Medium Eagle (39 mL, Life Technologies, #21010046) supplemented with
12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mM
GlutaMax (Life Technologies) and 5% heat-inactivated horse serum (Life
Technologies, 26050070). Slices were imaged using a Leica SP8 confocal
microscope. Approval for rodent experiments was obtained from the Stanford
University’s Administrative Panel on Laboratory Animal Care (APLAC).
Birey F., Andersen J., Makinson C.D., Islam S., Wei W., Huber N., Fan H.C., Cordes Metzler K.R., Panagiotakos G., Thom N., O’Rourke N.A., Steinmetz L.M., Bernstein J.A., Hallmayer J., Huguenard J.R, & Pașca S.P. (2017). Assembly of functionally integrated human forebrain spheroids. Nature, 545(7652), 54-59.
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Organotypic Forebrain Slice Culture Protocol

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Organotypic cultures of mouse coronal forebrain slices were prepared
following published methods45 (link)with some modifications. Whole brains from E14–E18 mouse embryos were
embedded in 4% low-melting point agarose and slices were cut at
250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100
ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30
ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1
M NaHCO3). Slices with visible forebrain structures were placed in membrane
inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated with
Poly-L-orthinine and Laminin (Sigma) overnight. They were cultured in a Basal
Medium Eagle (39 mL, Life Technologies, #21010046) supplemented with
12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mM
GlutaMax (Life Technologies) and 5% heat-inactivated horse serum (Life
Technologies, 26050070). Slices were imaged using a Leica SP8 confocal
microscope. Approval for rodent experiments was obtained from the Stanford
University’s Administrative Panel on Laboratory Animal Care (APLAC).
Birey F., Andersen J., Makinson C.D., Islam S., Wei W., Huber N., Fan H.C., Cordes Metzler K.R., Panagiotakos G., Thom N., O’Rourke N.A., Steinmetz L.M., Bernstein J.A., Hallmayer J., Huguenard J.R, & Pașca S.P. (2017). Assembly of functionally integrated human forebrain spheroids. Nature, 545(7652), 54-59.
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3

Culturing CFBE41o-ΔF cells for CFTR analysis

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CFBE41o-ΔF (CFBE41o- cells expressing ΔF508 CFTR) cells were routinely maintained in DMEM (Dulbecco’s modified Eagle’s medium) and Ham’s F12 medium (50:50, v/v; Life Technologies) at 37°C in a humidified incubator containing 5% CO2 as described previously [27 (link)]. For low-temperature and delivery of ΔF508-CFTR to the plasma membrane, cells were incubated at 27°C for 24 h or 48 h prior to experiments as indicated. For polarized monolayers, CFBE41o-ΔF cells were seeded onto membrane inserts (Costar, Corning) and cultured on an air-liquid interface for 4 to 5 days before analysis as described previously [10 (link)].
Fu L., Rab A., Tang L.P., Bebok Z., Rowe S.M., Bartoszewski R, & Collawn J.F. (2015). ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments. PLoS ONE, 10(4), e0123131.
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4

Organotypic Hippocampal Slice Cultures Aβ Treatment

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Organotypic hippocampal slice cultures (OHSC) were prepared from wild-type C57Bl6 mice at 7 days of age and maintained as described (Harris-White et al., 1998 ; Stoppini et al., 1991 (link); Teter et al., 1999 (link)). Briefly, 400 μm slices were maintained in membrane inserts (Costar, Cambridge, MA, 0.4 mm) using media of MEM plus HEPES with the serum substitute TCM (final concentration 2%; ICN Pharmaceuticals, Costa Mesa, CA). Starting on the first day in vitro (0 DIV), cultures were treated with Aβ40 (15 μg/ml) for 4 days. The medium was changed to that without Aβ and cultured for 4 more days, to allow Aβ deposits to develop. At 8 DIV, cultures were treated for 4 days with or without curcumin (at two doses), or the anti-Aβ antibody 10G4 (5 μg/ml), or control isotype antibody IgG2b. The media was changed at day 2 of the treatment period to fresh media containing the respective treatments. The cultures were harvested, extracted with formic acid, and Aβ measured by ELISA. Controls (not shown) were used to show that addition of 10G4 to cultures immediately before formic acid treatment did not interfere with the ELISA, which used 10G4 for detection, presumably because formic acid irreversibly denatured the 10G4 antibody.
Teter B., Morihara T., Lim G.P., Chu T., Jones M.R., Zuo X., Paul R.M., Frautschy S.A, & Cole G.M. (2019). Curcumin restores innate immune Alzheimer’s disease risk gene expression to ameliorate Alzheimer pathogenesis. Neurobiology of disease, 127, 432-448.
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5

Aflibercept Transcytosis Assays in iBREC

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Transcytosis assays were performed as published to assess transport of aflibercept through a confluent iBREC monolayer cultivated on membrane inserts (4.7 cm2, pore size 0.4 μm; Corning) from the lower to the upper chamber [7 (link), 12 (link)]. After incubating iBREC for 1 day in SHM or SFM, aflibercept (final concentration, 250 μg/ml) was placed in the bottom chamber and samples were then taken from the upper chamber at indicated time points. Presence of aflibercept in these samples was assessed by Western blot analyses and peak volumes of the corresponding bands (four replicates) determined with EvolutionCapt software (Vilbert Lourmat) were normalized in relation to those obtained from 1 ng aflibercept.
Deissler H.L., Lang G.K, & Lang G.E. (2018). Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation. Graefe's Archive for Clinical and Experimental Ophthalmology, 257(1), 83-94.
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6

Placenta-on-a-Chip: Microfluidic Device

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The placenta-on-a-chip
consists of two polydimethylsiloxane (PDMS)
slabs, each with a microchannel (Figure 1d) constrained by the following dimensions:
100 μm (height) and 400 μm (width). The microfluidic device
was fabricated using a silicon wafer SU-8 mold produced through standard
soft lithography techniques. A mixture of PDMS base and curing agent
solution (Dow Corning) at 10:1 (w/w) was introduced into a SU-8 mold
placed in a Petri dish (15 cm in diameter), as previously described.23 (link) The PDMS was cut and removed from the mold as
upper and lower layers after the PDMS had solidified at room temperature.
A biopsy punch was then used to produce inlet/outlet holes (1 mm in
diameter) on the upper PDMS layer. A polyethylene terephthalate (PETE)
membrane (0.4 μm pore size) as the barrier between two channels,
taken from the membrane inserts (Corning), was placed over the midsection
of the lower channel, after which both PDMS layers were plasma-treated,
aligned, bonded together, and left overnight to completely cure the
bond. The chip was UV-sterilized for 20 min following attachment of
inlet/outlet tubing to provide fluid access to each channel, as described
previously.23 (link)
Pemathilaka R.L., Alimoradi N., Reynolds D.E, & Hashemi N.N. (2022). Transport of Maternally Administered Pharmaceutical Agents Across the Placental Barrier In Vitro. ACS Applied Bio Materials, 5(5), 2273-2284.
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7

3D-Printed Drug Delivery Device for Spheroid Assay

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Spheroids were dosed with the use of a 3D printed device as discussed previously.24 (link) Briefly, spheroids were placed into membrane inserts (0.4 μm pore diameter; Corning Incorporated, Corning, NY, USA) that fit into the 3D printed device. These inserts rest on top of a flow channel, which allows for the diffusion of small molecules. The device has 6 identical channels, which can accommodate 6 experiments at once and allows for 36 biological replicates in a single run. The spheroids were dosed using the clinical timescale for the FOLFIRI treatment regime over a 24 hour period, depicted in Figure 1.46 (link),47 (link) First, 11.3 μM folinic acid was added to the inserts that housed the spheroids within the 3D printed device for the first hour. At the start of the second hour, 20.6 μM irinotecan was manually added to the inserts. At the start of the third hour, 68.5 μM 5-fluorouracil was manually added to the inserts and flowed through the 3D printed device. The top of the inserts was covered to prevent evaporation.
LaBonia G.J., Ludwig K.R., Mousseau C.B, & Hummon A.B. (2017). iTRAQ Quantitative Proteomic Profiling and MALDI-MSI of Colon Cancer Spheroids Treated with Combination Chemotherapies in a 3D Printed Fluidic Device. Analytical chemistry, 90(2), 1423-1430.
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8

Transwell Invasion Assay Protocol

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The invasion assay was performed in a transwell chamber consisting of a 24‐well plate with membrane inserts (Corning, New York, USA) containing 8‐mm‐pore‐sized polycarbonate filters precoated with 60 μl of 1 mg/ml Matrigel matrix solution (BD Biosciences, San Jose, USA). A suspension of 4 × 104 cells in 200 μl of serum‐free culture medium was added to the inserts, and each insert was placed in the lower chamber containing 600 μl of 10% fetal bovine serum (FBS) culture medium. After 24 h, the cells that penetrated the membrane were fixed with 4% paraformaldehyde and stained with crystal violet. Images were captured by using the Evos Fl Color Imaging System (Thermo Fisher Scientific, Waltham, USA).
Xiong L., Ye X., Chen Z., Fu H., Li S., Xu P., Yu J., Wen L., Gao R., Fu Y., Qi H., Kilby M.D., Saffery R., Baker P.N, & Tong C. (2021). Advanced Maternal Age‐associated SIRT1 Deficiency Compromises Trophoblast Epithelial−Mesenchymal Transition through an Increase in Vimentin Acetylation. Aging Cell, 20(10), e13491.
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9

Transwell Assay for Cell Migration and Invasion

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Transwell migration and invasion assays were conducted in 96-well plates with membrane inserts (8 µm pore-size) (Corning, New York, NY, USA). Cells were treated with 10 µM PFOS for 72 h. After that, 5 × 105 cells were resuspended in 50 µl of assay medium and seeded in the upper chamber of transwells with (invasion assay) or without (migration assay) Matrigel Matrix (200 µg/ml). The lower chamber contained 100 µl growth medium. Cells were incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO2. At the end of incubation, non-invasive cells in the upper chamber were removed and invasive cells in the bottom were fixed with 4% formaldehyde and stained with DAPI and then were counted as described in the Sect. “Cell counting by DAPI staining” of “Materials and methods”. Cells were viewed in an ImageXpress Micro XLS Widefield High-Content Analysis System (Molecular Devices, Sunnyvale, CA, USA), and images were analyzed with the SoftMax Pro Software after digital acquisition (Molecular Devices, Sunnyvale, CA, USA).
Pierozan P, & Karlsson O. (2017). PFOS induces proliferation, cell-cycle progression, and malignant phenotype in human breast epithelial cells. Archives of Toxicology, 92(2), 705-716.
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10

Transwell Invasion Assay for Cells

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Cell invasion capacity was assessed using transwell plates containing membrane inserts with 0.8-μm pores (Corning, USA). membrane inserts were coated with Matrigel (BD Biosciences) then complete culture medium (600 µL) was added to the lower chamber followed by insertion of membrane inserts into transwell plates. Next, 200 μL of cell suspension in serum-free medium (1 × 105 cells/mL) with or without GDNPs was added to the upper chamber. After 24 h of incubation under standard cell culture conditions, non-invasive cells were removed from the upper chamber with a cotton swab then remaining cells on membrane inserts were fixed in 4% PFA for 15 min at RT. Next, cells were stained with 0.1% crystal violet solution for 20 min. Cell invasion was visually assessed microscopically based on counting of cells in five random fields per well.
Yang L., Jin W.Q., Tang X.L., Zhang S., Ma R., Zhao D.Q, & Sun L.W. (2022). Ginseng-derived nanoparticles inhibit lung cancer cell epithelial mesenchymal transition by repressing pentose phosphate pathway activity. Frontiers in Oncology, 12, 942020.
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