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Transwell inserts with 8 μm pores

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Transwell inserts with 8 μm pores are a laboratory equipment component used in cell culture studies. These inserts facilitate the examination of cell migration and invasion through a porous membrane. The 8 μm pore size allows for the passage of cells while maintaining a barrier between the upper and lower chambers.

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Lab products found in correlation

Axioskop 2 plus, by Zeiss (4 mentions) Axiovision microscope, by Zeiss (2 mentions) Blebbistatin, by Bio-Techne (1 mentions) Rs504393, by Bio-Techne (1 mentions) H1152, by Bio-Techne (1 mentions) Celltiter 96 aqueous one solution proliferation kit, by Promega (1 mentions) Matrigel, by BD (1 mentions) Nucblue fixed cell stain, by Thermo Fisher Scientific (1 mentions) Maraviroc, by R&D Systems (1 mentions) Anti cd31 antibody, by Thermo Fisher Scientific (1 mentions) Collagenase a, by Roche (1 mentions) Anti rat igg microbeads, by Miltenyi Biotec (1 mentions) Matrigel matrix, by BD (1 mentions)

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Transwell inserts (350 citations) 8-μm pores (8 citations)

13 protocols using transwell inserts with 8 μm pores

1

Transwell Assay for VEGF-Stimulated Cell Migration

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RF/6A cells suspended at 1 × 104 cells/200 μL were seeded on fibronectin coated Transwell inserts with 8 μm pores (BD Biosciences) pre-coated with 3% bovine serum albumin, then stimulated with 10 ng/mL of VEGF in the lower chamber. After 4 h, the Transwell inserts were fixed in 4% PFA and stained with 1% crystal violet. Digital microscopic images of the underside of each Transwell insert were taken at three independent microscopic fields per transwell.
Hayashi H., Mamun A.A., Takeyama M., Yamamura A., Zako M., Yagasaki R., Nakahara T., Kamei M, & Sato M. (2019). Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization. Scientific Reports, 9, 1560.
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2

Lung Endothelial Cell Transmigration Assay

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Primary lung endothelial cells (3x104) were seeded on gelatin-coated 24-well transwell inserts with 8μm pores (BD) and allowed to grow to confluency. Endothelial cells were pre-incubated with the following inhibitors: 50 μM RS504393 (CCR2), 50 μM 2-APB (IP3-gated Ca+ channels), 10 μM BAPTA-AM (chelating intracellular Ca2+), 1 μM ML-7 (MLCK), 5 μM H-1152 (ROCK2), 2.5 μM Blebbistatin (myosin II ATPase) for two hours in RPMI1640/10% FCS medium (all inhibitors were purchased from Tocris). The first four inhibitors were used according to manufacturer’s recommendations (Tocris); for H-1152 and Blebbistatin we used concentrations as previously published (28 (link)). Upon removal of inhibitors from the endothelial cells MC-38GFP cells (2x104) were added in presence or absence of CD115+ monocytes (1x105) in RPMI1640/3% FCS. Transmigration was induced with RPMI1640/10% FCS in the lower chamber and terminated after 16 hours. Alternatively, MC-38GFP cells (2x104) were added to the upper insert with or without rhCCL2 (1 μg/ml; kindly provided by A. Kungl, University of Graz). The number of transmigrated MC-38GFP cells was counted using a Zeiss AxioVision microscope (n ≥ 3).
Roblek M., Protsyuk D., Becker P.F., Stefanescu C., Gorzelanny C., Glaus Garzon J.F., Knopfova L., Heikenwalder M., Luckow B., Schneider S.W, & Borsig L. (2018). CCL2 is a vascular permeability factor inducing CCR2-dependent endothelial retraction during lung metastasis. Molecular cancer research : MCR, 17(3), 783-793.
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3

Transwell Invasion and Proliferation Assay

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Transwell inserts with 8-μm pores (BD Biosciences, San Jose, CA, USA) were coated with Matrigel (BD Biosciences). Approximately, 4 × 104 cells were seeded on Matrigel-coated upper chambers with 0.3 ml serum-free media, and 0.5 ml of medium with 10% FBS was placed in the lower wells. The invaded cells were fixed and stained in 25% methanol and 0.5% crystal violet after 48-h incubation. Proliferation was determined by using the Celltiter96AQueous One Solution Proliferation Kit (Promega, Madison, WI, USA). Invasion was assessed as the number of cells that had invaded through the membrane normalized by the proliferation.
Alesi G., Jin L., Li D., Magliocca K., Kang Y., Chen Z., Shin D., Khuri F, & Kang S. (2016). RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis. Oncogene, 35(41), 5412-5421.
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4

Macrophage Differentiation and Migration Assay

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Macrophages were differentiated from BM cells (after a dmPGE2 pulse or after isolation from chimeric mice) as described previously [7 (link)–9 ]. Briefly, BM cells were cultured in RPMI-10 and 0.6 ng/ mL (50 IU/mL) CSF-1 (gift from Dr. E.R. Stanley) for 24 h. Nonadherent cells were then collected and cultured with 12 ng/ mL CSF-1 for 3 days (replated on day 2), followed by 5 days with 120 ng/mL CSF-1 before use. Adherent cells were uplifted using PBS containing 2 mmol/L ethylenediaminetetraacetic acid (EDTA). BM-differentiated macrophages (2.5 × 105) were seeded, in replicate, into Transwell inserts with 8 μm pores (BD Biosciences) in 200 μL of RPMI-10 (CSF-1-free or CCL-2-free) and inserts were placed into a 24-well companion plate with complete medium containing 120 ng/mL CSF-1 or 20 ng/mL CCL2, respectively. Although CSF-1 is necessary for macrophage growth and differentiation, it is also a potent chemokine that stimulates macrophage migration [8 (link),9 ]. CCL2 is a macrophage chemoattractant with a recognized ability to recruit monocytes to sites of inflammation [10 ]. The macrophages migrated for 5 hours (37°C, 5% CO2) before fixation of inserts in 4% paraformaldehyde, staining for 5 min with NucBlue-fixed cell stain (Life Technologies, Australia), and imaging by confocal microscopy. Migrated cells were counted for 10 representative fields per insert at 20 × magnification.
McGonigle T.A., Dwyer A.R., Greenland E.L., Scott N.M., Keane K.N., Newsholme P., Goodridge H.S., Zon L.I., Pixley F.J, & Hart P.H. (2017). PGE2 pulsing of murine bone marrow cells reduces migration of daughter monocytes/macrophages in vitro and in vivo. Experimental hematology, 56, 64-68.
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5

Cell Invasion Assay Using Transwell

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The cell invasion assay was carried out using Transwell inserts with 8‐μm pores (BD Biosciences, San Jose, CA, USA), based on the manufacturer's protocol and previous reports.26, 27 Cells were seeded into inserts for 24‐well plates at 4 × 104 cells per insert in serum‐free medium and then transferred to wells filled with the culture medium containing 10% FBS. After 24 h incubation, non‐invading cells on the top of the membrane were removed by cotton swabs. The invading cells were counted using a microscope in five random visual fields (×200 magnification).
Yamamoto M., Takahashi T., Serada S., Sugase T., Tanaka K., Miyazaki Y., Makino T., Kurokawa Y., Yamasaki M., Nakajima K., Takiguchi S., Naka T., Mori M, & Doki Y. (2017). Overexpression of leucine‐rich α2‐glycoprotein‐1 is a prognostic marker and enhances tumor migration in gastric cancer. Cancer Science, 108(10), 2052-2060.
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6

Quantifying Cell Migration Dynamics

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Cells were seeded onto 6-well plates. The monolayer was scratched with a 10 μl pipette tip and then incubated in serum-free RPMI-1640 medium. Images of the wells were taken at different time points (0, 24, and 48 h) on an inverted microscope (Axioskop 2 Plus; Zeiss, Germany). The length of the open area was calculated with software. Cell migration was evaluated using Transwell inserts with 8 μm pores (BD Biosciences, San Jose, CA, USA). Briefly, cells were released using trypsin-EDTA and sequentially rinsed with RPMI-1640 medium containing 10% FBS. The rinsed cells were resuspended in serum-free RPMI-1640 medium, and 200 μl of the cell suspension (1 × 105 cells) was added to the Transwell insert chamber with a filter that was coated with Matrigel. In the lower chamber, 500 μl of RPMI-1640 medium containing 10% FBS was added as a chemoattractant. After 24 h of incubation, the cells in the lower chamber were fixed, stained with hematoxylin and counted under a microscope.
Chen B., Wang S.Q., Huang J., Xu W., Lv H., Nie C., Wang J., Zhao H., Liu Y., Li J., Lu C., Zhang J, & Chen X.B. (2021). Knockdown of Kremen2 Inhibits Tumor Growth and Migration in Gastric Cancer. Frontiers in Oncology, 10, 534095.
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7

Transwell Invasion Assay Protocol

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Invasion assays were performed in trans-well inserts with 8 μm pores (BD Biosciences) coated with 20% growth-factor-reduced Matrigel. 2 × 105 cells were seeded into the upper chamber/per well in serum-free medium, and the lower chambers filled with complete media as a chemo-attractant. The chambers were incubated for 24 h at 37 °C with 5% CO2. Migrated cells on the undersides of filter membrane were fixed in 4% paraformaldehyde and washed three times in PBS. Then, the migrated cells were stained with crystal violet and counted using light microscopy. Experiments were performed in triplicate.
Wang L., Sun J., Yin Y., Sun Y., Ma J., Zhou R., Chang X., Li D., Yao Z., Tian S., Zhang K., Liu Z, & Ma Z. (2021). Transcriptional coregualtor NUPR1 maintains tamoxifen resistance in breast cancer cells. Cell Death & Disease, 12(2), 149.
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8

Transwell Invasion Assay Protocol

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In brief, transwell inserts with 8-μm pores (BD Biosciences) were coated with Matrigel (272 μg/ml). Approximately 4 × 105 cells were seeded in the upper chambers in 500 μl serum free medium, while 500 μl of medium supplemented with 10% FBS as a chemoattractant was placed in the lower wells. The chambers were incubated at 37°C in a CO2 incubator. After 48 hours, the chambers were pulled out, and the non-invading cells on the upper surface were removed with the cotton swab. The cells that invaded to the lower surface of the membrane were fixed in methanol, air dried, and stained with 0.1% crystal violet for 10 minutes. The stained cells were counted in 10 random fields (at ×200 magnification) using a light microscope. Fold invasion was calculated as the number of cells that had passed through the Matrigel-coated membranes relative to the untreated or empty control transfected cells.
Peres J., Mowla S, & Prince S. (2014). The T-box transcription factor, TBX3, is a key substrate of AKT3 in melanomagenesis. Oncotarget, 6(3), 1821-1833.
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9

Quantifying Cell Migration and Invasion

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Roles of miR-99a, miR-99b, and miR-100 in the cell migration and invasion abilities were measured using transwell inserts with 8 μm pores (BD Biosciences, San Jose, CA, USA). For migration assay, 2×105 cells were added into the upper chamber with a noncoated membrane. For invasion assay, 2×105 cells were added into the upper chamber precoated with matrigel matrix. Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum was added into the lower chambers. After overnight incubation, cells adhering to the upper surface of the membrane were removed and the bottom (migrated or invasive) cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet to visualize nuclei. The number of migrated or invasive cells was counted under a microscope in five random fields and the means for each chamber were determined. All experiments were repeated three times and data were obtained from three independent experiments.
Zhang M., Guo Y., Wu J., Chen F., Dai Z., Fan S., Li P, & Song T. (2016). Roles of microRNA-99 family in human glioma. OncoTargets and therapy, 9, 3613-3619.
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10

Cell Invasion Assay Using Transwell

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The cell invasion assay was performed using transwell inserts with 8 μm pores (BD Biosciences, San Jose, CA, USA), in accordance with the manufacturer’s protocol. The invading cells were counted using a microscope in three random visual fields (×200 magnification).
Mokutani Y., Uemura M., Munakata K., Okuzaki D., Haraguchi N., Takahashi H., Nishimura J., Hata T., Murata K., Takemasa I., Mizushima T., Doki Y., Mori M, & Yamamoto H. (2016). Down-Regulation of microRNA-132 is Associated with Poor Prognosis of Colorectal Cancer. Annals of Surgical Oncology, 23(Suppl 5), 599-608.
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