Millicell® cell culture inserts provide a permeable support for cell growth, allowing cells to access media and nutrients from the apical and basolateral sides. This feature more closely promotes an in vivo cellular environment for cells than conventional seeding on polystyrene plates, enabling researchers to perform a wide variety of cellular assays and helping assess many facets of biology.
The optically clear and low pore density polyethylene terephthalate (PET) membrane in Millicell® hanging cell culture inserts allows for cell monitoring during cell growth, ensuring accurate seeding, proliferation, and morphology during experimentation. Clear membrane inserts can also be utilized with various staining tools for bioimaging such as small molecule fluorophores and fluorescent probes, antibody-based techniques, and histological staining.
The clear membrane allows researchers to image cells directly on the insert after staining. This offers a convenient and easier way to perform post-processing and observe cell growth and membrane integrity, specific cellular features, and determine cellular differentiation. Millicell® hanging cell culture inserts also support common quantitative barrier assays such as transepithelial-transepithelial electrical resistance (TEER) measurements and Lucifer Yellow assay in addition to easy visualization.
This article describes how to prepare inserts for fluorescence and immunofluorescence staining and microscopic analysis directly from the insert. We also demonstrate how to utilize imaging to support quantitative barrier measurements.
Figure 1: Millicell® clear 1.0 μm inserts (PTRP24H48) containing no cells or seeded with MDCK II cells. Membranes were fixed and stained in the insert. Inserts were placed on a 12-well glass plate in PBS and imaged for nucleus (DAPI), tight junction proteins (ZO-1), and actin (phalloidin) under a fluorescence microscope.
Figure 2: MDCK II cells grown on Millicell® 1.0 μm clear inserts (PTRP24H48) over 7 days. Cell growth was monitored directly in the insert using the Millicell® Digital Cell Imager. By Day 5, MDCK II cells formed a cobblestone-like morphology indicative of barrier formation.
The optically clear membrane in the Millicell® cell culture inserts allows fluorescent imaging of MDCK II cells directly from the cell culture plate. This bypasses the need to excise the membrane from the housing, optimizing the workflow. Below are optimal method(s) to image the insert from the plate.
Image directly from cell culture plate (same well-size)
Note: Decreasing the objective working distance can impact the clarity of the imaging. Utilize well plates with shorter well height, which allows the insert and membrane to sit closer to the bottom of the well during imaging. Glass well bottom plates from MatTek can increase the clarity of fluorescence images, notably for images taken at higher objectives.
Image directly from cell culture plate (larger well-size)
Figure 3. MDCK II cells grown on Millicell® 1.0 μm clear inserts (PTRP24H48). Cells were stained and imaged directly in insert with different cell culture plates. Greiner Bio plates have a short well height, which decreases the objective working distance and increases clarity as compared to competitor standard plates. The MatTek glass well bottom plates increase the clarity of fluorescence images.
Note: If excising the membrane is required, we recommend using a no. 11 scalpel to slowly excise membrane at the outer circular edge of the housing. A no. 13 scalpel can then be used to trim or divide membrane into separate pieces for staining.
Figure 4. Millicell® inserts can be stored at 4°C under two conditions. After staining, the insert can be stored in (A) a 24-well plate with PBS and (B) in antifade solution. Fluorescence images are taken after one week of respective preservation.
Millicell® cell culture inserts provide robust permeable support for cell growth and proliferation and are ideal for common quantitative barrier assessments such as TEER and Lucifer Yellow assays. The clear membrane allows users to easily connect these quantitative measurements with cellular visualization of the barrier.
For more information and sample protocols on barrier formation and permeability assays, visit our technical article page.
We show that visualization of cellular features using Millicell® 1.0 μm clear inserts supports the quantitative measurements assessed from Lucifer Yellow and TEER assays in a barrier formation assay. We also demonstrate there is no significant difference between Millicell® clear 1.0 µm hanging insert versus competitor clear 1.0 µm hanging insert.
Figure 5. Millicell® 1.0 μm clear inserts (PTRP24H48) were initially seeded with three different densities. On Day 3, barrier permeability and integrity were measured using Lucifer Yellow assay and TEER. Inserts were stained for the tight junction protein ZO-1 to visualize the barrier. The data demonstrate that lower seeding densities have slower barrier formation and better passthrough when compared to the lower TEER values seen at higher seeding densities.
Figure 6. Time course of MDCK II cells (initial seeding: 1250 cells/insert) growing on a clear Millicell® insert (PTRP24H48). Cells were fixed and stained directly in insert for nucleus (DAPI), tight junction proteins (ZO-1), and actin (phalloidin). Inserts were imaged directly in a 12-well glass bottom plate under fluorescence microscope. Qualitative assessment would suggest that barrier integrity and permeability are at peak on Day 5 due to the compact nucleus formation and robust ZO-1 staining.
Figure 7. Barrier permeability assessed by Lucifer Yellow assay. MDCK II cells were grown on clear Millicell® inserts (PTRP24H48) and two competitor (Competitor S and Competitor F) clear 1.0 µm inserts. Inserts were tested on Days 3, 5, and 7. By Day 5, the data demonstrates cells grown on the Millicell® insert form a robust barrier, with <5% LY passthrough, similar to competitor inserts.
Figure 8. Barrier integrity assessed by TEER. MDCK II cells were grown on clear Millicell® inserts (PTRP24H48) and two competitor (Competitor S and Competitor F) clear 1.0 µm inserts. Inserts were tested on Days 3, 5, and 7. By Day 5 the data demonstrates cells grown on the Millicell® inserts form a robust barrier and stabilize by Day 7 (≥50 ꭥ·cm2), similar to competitor inserts.
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