6 Cell Culture Inserts in 6 Well Plate, 0.4μm PC pore size 4.1cm² Surface, Pack of 6 - FAQs

What is the difference between the 24-well plate format for the Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates (Cat. No. 140620) and the 24-well plate format for the Nunc Cell Culture Inserts in Carrier Plate Systems (Cat. No. 141002)?

The difference between the two 24-well plate formats is as follows:

• Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates (Cat. No. 140620): There are only 12 inserts in the 24-well multi-well plate, leaving every second column free without an insert. This is a preferred configuration for some co-culture experiments, as it makes it easy to access the culture grown in the multi-well plate for media changes, etc., by placing the insert in the empty well next to it. The inserts are placed directly into the wells here, leaving a 0.9 mm gap between the bottom of the well and the insert membrane.
• Nunc Cell Culture Inserts in Carrier Plate Systems (Cat. No. 141002): There are 24 inserts in the 24-well carrier plate. The carrier plate with the hanging slots allows for adjustment of the insert height. The three hanging positions in the carrier plate allow for distance of approximately 0.9 mm, 3.3 mm, or 6.3 mm between the well-bottom and insert growth surface for both 12- and 24-well plate formats. This configuration is preferred when customer needs more versatility, for example to facilitate air-liquid interface culture, and other co-culture applications.

With the Nunc Polycarbonate Cell Culture Inserts in Multi-Well Plates, what is the distance between the cell culture membrane and the bottom of the multi-well dish?

The membrane is approximately 1.0 mm above the bottom of the dish for the Nunc Polycarbonate Cell Culture Inserts.

Can I observe cells on the cell culture membrane when the Nunc Polycarbonate Cell Culture Insert is inside of a multi-well plate?

Due to the large pore size of the Nunc polycarbonate inserts, spots or holes are observed and they represent the membrane. In this plane of focus, the most visible cells are the ones located on the membrane, whereas the slightly blurred backdrop are the cells on the dish that are in a different plane of focus. These cells do not photograph with great resolution due to the slightly opaque quality of the membrane. However, these cells are visible in culture.

What is the recommended Multidish chamber media volume per carrier plate insert and height position?

Low position: 2 mL (12-well), 1 mL (24-well), Medium position: 3 mL (12-well), 1.5 mL (24-well), High position: 4 mL (12-well), 2 mL (24-well). Some optimization may be necessary for development purposes.

Why are only 12 inserts provided with standard membrane insert products in the 24-well format?

This has emerged as the industry standard. In many cases having empty wells facilitates media changes and other experimental steps.

What is the difference between your cell culture membrane inserts and carrier plates?

Standard culture membrane inserts are placed directly into the wells of a Multidish, and the feet at the bottom of the insert maintain a distance of about 1 mm between the membrane and the Multidish culture surface. The “carrier plate” culture membrane product adds a tray between the Multidish and the lid, with a set of hanger brackets at each well. These brackets engage one of three sets of tabs on each insert, to allow for selection between 3 different distances (approximately 1, 3, or 6 mm) between the membrane and the Multidish culture surface. This can allow for more flexibility in experiments, such as distance-dependent interaction between populations or media volume under cultures at the air-media interface.

Are POROS analytical columns compatible with HPLC systems from Waters or Agilent?

All of the pre-packed POROS columns, regardless of column size or resin, have the same 10-32 female fittings on the column. Columns are compatible with Waters and Agilent HPLC systems. You will need 1/16 inch male fittings for the HPLC to attach the column.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

For any POROS analytical column, is the column compatible with an AKTA or Avant purification system?

All of the pre-packed columns, regardless column size or resin, have the same 10-32 female fittings on the column. They are all compatible with the AKTA instrument platform, however due to the column volume of the 2.1 mmD x 30 mmL columns (0.1 mL), these will not perform well in AKTA or Avant purification systems due to the relative size of the hold- up volume in these systems. The smallest column (PEEK or SS) that could feasibly be used in these systems is the 4.6mmD x 50mmL (0.8 mL) format.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Are the pre-packed POROS CaptureSelect or any POROS analytical columns compatible with an UPLC or is the use of these columns limited to HPLC?

UPLC is typically described as chromatography with sub-2 µm particles, which drives higher efficiency/better separation independent of operating flow rate. As particle size decreases, backpressure increases so the UPLC systems tend to have higher pressure ratings compared to HPLC systems. The flow rates on UPLC systems would be sufficient for POROS columns. Pump capabilities are typically 0.1-1.0 mL/min, so not as fast as a HPLC, but sufficient. Hold-up volumes on UPLC systems are lower than HPLCs, so this can help with sensitivity and efficiency. So from a pressure and flow standpoint there should be no challenge to operate a POROS CaptureSelect column on a UPLC system.

That being said, care should be taken running POROS columns on UPLC systems. Higher pressure safety interlock MUST be set at the rated column pressure, typically 180 bar. HPLC column hardware is not rated to operate at the static pressures possible and typical on UPLC systems. A clog or the like on a UPLC system could generate unsafe pressures on any HPLC-rated column before the default UPLC safety pressure interlock is tripped.

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What is the physical or mechanical stability of POROS resins?

The physical stability of POROS resins is high. Customers have agitated, with overhead mixing and or bottom gassing for long periods of time (5-10 hours) and have performed hundreds of diaphragm pump passes during stall packs. Keep in mind that agitation with any device that creates grinding and sheer, such as with a magnetic stir bar, is not recommended for any type of resin.

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What should I do if a POROS column dries out or air is pumped onto the column?

POROS resins rehydrate quite well. Here is the procedure that we recommend:

1. Flow 5% ethanol over the column at ?350 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
2. If baseline noise is still present, flow 20% ethanol over the column at 300 cm/hour for 3 CVs, followed by water or 0.1 M sodium chloride at 300 cm/hour for 3 CVs.
3. If you have established column qualification tests and specifications, perform the tests after rewetting.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Can POROS resins be used to purify large biomolecules?

In general POROS resins have behaved better for large biomolecules such as viral particles, fusion proteins, and globular proteins, where shape (not size) matters due to its unique pore structure and large pore structure as compared to traditional resins. As the target molecule size increases, capacities obtained will decrease. The large pore structure of POROS resins, which allows for convective flow (and therefore enhanced diffusion) is especially well suited for the purification of large biomolecules. POROS resins offer the best blend of high capacity (associated with chromatography beads) and improved chromatography efficiency (typically associated with monoliths or membranes). The average pore size for POROS resins is 1,000-3,600 Angstrom (100-360 nm) depending on the base bead/chemistry being utilized. For example, the molecular weight of IgG is 150 kDa and the molecular radius is 55 Angstrom. The molecular weight of IgM is ~900 kDa (pentamer), and the molecular radius is 120 Angstrom. So both of these biomolecules can interact with the pore structure associated with POROS materials.

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How many uses should I expect out of my POROS or CaptureSelect column?

Resin lifetime depends on how the resin is used and the cleaning process that is employed. Therefore, each purification process will need to be evaluated specifically, especially if long lifetime is desired. Because binding can be different between resins, different cleaning schemes may be needed. An unoptimized cleaning process of any resin may yield <5 uses, and an optimized cleaning process can yield a hundred cycles or more. Discoloration of resin can occur for different reasons from process compounds such as metals like iron (Fe2+/3+) and copper (Cu2+), additives to the media (like vitamin B12 and folic acid), and elution solutions (like magnesium chloride, Mg2+). We can provide recommendations that have proved helpful for many customers to optimize the life of their POROS or CaptureSelect column.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

What type of column do you recommend for POROS 50 µm resins? Do I need a high pressure column for POROS resins?

POROS resins are mechanically rigid and incompressible and can be packed effectively in low-pressure glass columns and in high-pressure stainless steel columns. The lack of wall support with increasing column diameter has minimal impact on chromatography performance because the beads support themselves, allowing for flexible column packing approaches and consistent and robust results. Columns can be packed with traditional flow pack, axial compression, or pack-in-place/stall pack packing methods.

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What are the benefits of POROS chromatography resins in a manufacturing process?

Increased titers and product demand have caused substantial bottlenecks in downstream processing for a range of biomolecules. POROS chromatography resins address these challenges with solutions that help maintain performance and add process flexibility. Due to the polymeric nature of the backbone and the way we attach the ligands, the beads have very robust physical and chemical stability. This allows for aggressive solutions to be used, if needed, to improve cleanability and thereby increase resin lifetime/reuse. Our ion exchange resins are the go-to resins in downstream monoclonal antibody and recombinant protein chromatography where capacity, resolution, and yield are critical. We are also working on a new line of hydrophobic interaction resins that may be of interest for future projects that will be a best-in-class product for capacity and resolution with an added benefit of being able to load under lower salt concentrations and faster flow rates. We are also a strong partner when it comes to quality and supply of our products.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.