Guidelines for using BD Falcon™ Cell Culture Inserts

Introduction
Directions for Use
Applications
Technical Bulletins available from BD Biosciences Discovery Labware

Introduction

BD Biosciences Discovery Labware offers a broad line of cell culture inserts incorporating polyethylene terephthalate (PET) track-etched membranes. Perfectly transparent, low pore density PET membranes provide a durable substrate for light microscopy, electron microscopy and immunofluorescence. These exceptionally strong membranes can be removed for staining, fixing or other procedures. Once removed, the membrane will not curl and remains easy to handle.

High pore density (HD) translucent PET membranes are more highly permeable substrates. They allow increased rates of basolateral diffusion of nutrients and molecules of interest for transport, secretion or binding studies.

Track-etched membranes have symmetrical, cylindrical pores. Both sides of the membrane are tissue culture treated and are suitable for cell growth.

Refer to the complete listing of BD Falcon™ Cell Culture Inserts below.

For information about our BD BioCoat™ extracellular matrix and matrix component inserts, please call 800.343.2035 or 978.901.7300 (in North America) or email us at labware@bd.com.

Catalog #	Membrane Material	Pore Size (microns)	Pore Density (pores/cm2)	Optical Quality	TC Plate (# wells)
353090	PET	0.4	1.6x106	Transparent	6
353180	PET	0.4	1.6x106	Transparent	12
353095	PET	0.4	1.6x106	Transparent	24
353102	PET	1.0	1.6x106	Transparent	6
353103	PET	1.0	1.6x106	Transparent	12
353104	PET	1.0	1.6x106	Transparent	24
353091	PET	3.0	8.0x105	Transparent	6
353181	PET	3.0	8.0x105	Transparent	12
353096	PET	3.0	8.0x105	Transparent	24
353093	PET	8.0	1.0x105	Translucent	6
353182	PET	8.0	1.0x105	Translucent	12
353097	PET	8.0	1.0x105	Translucent	24
353493	PET	0.4HD	1.0x108	Translucent	6
353494	PET	0.4HD	1.0x108	Translucent	12
353495	PET	0.4HD	1.0x108	Translucent	24
353092	PET	3.0HD	2.0x106	Translucent	6
353292	PET	3.0HD	2.0x106	Translucent	12
353492	PET	3.0HD	2.0x106	Translucent	24

NOTES:

All products are sterilized by gamma irradiation and are intended for single use only. HD signifies a high pore density membrane for maximum permeability. PET is tissue culture treated polyethylene terehthalate. PC is tissue culture treated polycarbonate.

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Handle inserts under aseptic conditions.
1.	Add prewarmed culture medium to each well of a Multiwell™ tissue culture plate. Refer to Table 2 for recommended working volumes.
2.	Aseptically, open the package, remove an insert with sterile forceps, and gently place it into the well. Avoid trapping air under the insert by tilting the insert while lowering it into the well.
	If you use BD Falcon™ Cell Culture Insert Companion Plates, position inserts with flanges resting in the notches on the top edge of each well. This will position inserts diagonally as show in Diagram A (below).
BD Falcon Cell Culture Insert Companion Plates have been specially designed for use with BD Falcon or BD BioCoat™ Cell Culture Inserts so that evaporation and contamination due to improper lid fit is eliminated. In the "Feeding Position" pipet access is improved for fluid handling on the basolateral side. Reagents can be added quickly and consistently for timed experiments. Aspiration of media from the well is easier, reducing the risk of contamination. In the "Incubation Position" BD Falcon Cell Culture Inserts remain locked in position in their Companion Plate wells. Media will not wick up between the insert and well wall. The patented BD Falcon low-evaporation lid provides a tortuous air-passage system that reduces evaporation and contamination. SIDE VIEW
3.	Seeding
3a.	It is recommended to store cell culture medium in incubator for 20 min. before seeding for pH equilibrium.
3b.	Add cells and media to the insert, referring to Table 2 for recommended working volumes. To determine the optimal seeding density for your cell type on a porous growing surface, we recommend using a range of seeding densities (cells/sq. cm) that brackets the seeding density used on nonporous surfaces (flasks, dishes and plates). For example: if you currently seed at 105 cells per sq. cm, seed at 0.5x105, 105 and 5x105 to determine the optimal initial seeding density. Refer to Table 2 for surface areas of inserts and wells.
Table 2: BD Falcon™ Cell Culture Inserts and Companion Plates Physical Specifications   6 Well 12 Well 24 Well Effective Diameter of Membrane (mm) 23.1 10.5 6.4 Effective Growth Area of Membrane (cm2) 4.2 0.9 0.3 Insert Height (mm) 17.2 17.2 17.5 Distance From Membrane to The Bottom of Well (mm) 0.9 0.9 0.8 Suggested Media In Insert (ml) 1.5-2.5 0.4-1.0 0.2-0.35 Suggested Media In Well (ml) 2.7-3.2 1.4-2.3 0.7-0.9 Insert Case Quality 48 48 48 BD Falcon™ Companion Tissue Culture Plate Catalog Number 353502 353503 353504 Growth Area In TC Plate Well (cm2) 9.6 3.8 2.0 Companion Plate Case Quantity 50 50 50 Companion Plate Total Volume (ml) 17.3 7.0 3.6
4.	Initial Attachment and Cell Culture Culture your cells under routine conditions. For some cells, initial attachment and lag phase may vary with insert material, pore size and pore density. After initial attachment, growth rates (doubling times) will generally be quivalent with equivalent times to confluency.
5.	Microscopy If you use transparent, low pore density membranes you can observe your live cultures using routine phase contrast or bright field microscopy. Large pore size and high pore density membranes may appear "speckled" due to shadows being cast by the pores.
6.	Feeding Use a standard 1 ml or pasteur pipet to remove media from above and below the membrane. If you use BD Falcon™ Cell Culture Insert Companion Plates, first slide inserts to one side as shown in Diagram A using a sterile pipet or forceps. This will provide better pipet access for aspirating and replacing media. Replace media with appropriate size pipet. Reposition inserts in the notches for incubation. Use of BD Falcon™ Cell Culture Insert Companion Plates may allow you to use a larger diameter (volume) pipet when dispensing media.
7.	Retrieving Cells To remove cells, follow your standard trypsinization or scraping procedure. Smaller diameter inserts can be scraped with a small rubber policeman or the blunt end of a Pasteur pipet. Note: When using larger pore size membranes, some liquid may drip through the membrane. This should be considered during trypsinization.
8.	Fixing and Staining Cells can be fixed using standard techniques. Inserts can be processed intact, by passing them through a series of fixation solutions. The membrane can easily be removed from the housing by cutting with a razor blade or scalpel to prepare sections for embedding, sectioning or staining. Inserts are stable under most processing conditions, and are recommended for TEM and SEM as described in BD Falcon™ Technical Bulletins No. 405 and 406.
9.	Extracellular Matrix The use of extracellular matrix proteins with porous supports provides a highly relevant in vitro model. A full line of matrix proteins, BD BioCoat™ precoated growth vessels and BD BioCoat™ precoated cell culture inserts is available from BD Biosciences Discovery Labware. For information about these products, please call 800.343.2035 or 978.901.7493 (Outside the United States).

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Applications

Membrane Type	Membrane Characteristics	Typical Applications
0.4 micron PET 3.0 micron PET	Strong Transparent Will not curl when removed from housing	Visualization by light microscopy Transmission and scanning electron microscopy Useful for studying transport of larger molecules (lipoproteins) and viruses Transendothelial migration Smooth muscle migration Endothelial cell migration
0.4 micron high pore density (HD)PET	Translucent Porosity equal to competitive 0.4 µm track-etched polycarbonate membranes High rates of basolateral diffusion	Transport, diffusion, and secretion of small molecules into, out of, or through a cell monolayer Barrier function (Transepithelial Electrical Resistance [TEER]) measurements Drug bioavailability assays
1.0 micron PET	Transparent Porosity and permeability equivalent to 0.4 HD membranes Good basolateral feeding, diffusion	General-purpose membrane Growth and visualization of live cells Transport, secretion, and diffusion of most molecules into, out of, and through cell monolayers Immunocytochemical staining Drug bioavailability assays In general, this is the maximum pore size available to prevent cell migration through pores
3.0 micron high pore density (HD) PET	High porosity, high permeability Rapid diffusion of large molecules, such as lipoproteins, virus and bacteria High rates of basolateral diffusion	Transport, secretion and diffusion of large molecules or viruses Cell migration studies This pore size offers maximum diffusion of large molecules or viruses
8.0 micron PET	High porosity, high permeability Large pores allow passage of mammalian cells	Tumor invasion Cell migration Chemotaxis Metastasis

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Technical Bulletins