Frequently Asked Questions

We offer a wide variety of chambers for cell migration assays. The following will help you determine your best options.

Which chamber should I choose?

Which filters should I use?

What Neuro Probe accessories should I buy?

Do I need coated filters for a migration assay?

How can I coat the filters?

How should I place the filter in my chamber?

How many cells should I load? At what concentration?

What if I am seeing no migrated cells?

Will I get crosstalk in my fluorescence reader using your clear microplates?

What is a “Boyden Chamber”?

Which chamber should I choose?

  1. What rate of throughput do you want to achieve?
  2. What are your budget considerations?
  3. How do you plan to quantify the results?

1. What rate of throughput do you want to achieve?

2. What are your budget considerations?

3. How do you plan to quantify the results?

  • When automating the counting process by using a microplate reader (fluorescence-type or ELISA-type), we recommend our disposable ChemoTx® 96-well system and the MB-series reusable 96-well microplate chambers, .
  • If you plan to use an ELISA-type reader, we recommend either the 116-series of our disposable ChemoTx® 96-well system or our reusable MBA96 chamber. There are two primary reasons for this. An ELISA-type detection system typically requires a relatively high minimum signal strength. To help maximize the signal, both of the products above offer a large exposed filter area. In addition, the larger well volume of the microplates used in these two systems facilitates the staining process required for many densitometric or colorimetric assays (e.g., MTT).
  • When working with non-adherent, or lightly adherent cells, we recommend our disposable ChemoTx® 96-well system or one of our reusable MB-series 96-well chambers to facilitate the collection of migrated cells.
  • For other types of microplate readers, e.g., luminescence readers or scintillation counters, see our page on Automated Readers.
  • To transfer migrated cells into the 96-well plate of your choice, we recommend our 96-well funnel plate (stock # FP1).
  • When visually quantifying cells you may use any of our migration instruments. Many researchers select our disposable ChemoTx® 96-well system for its low initial cost and ease of use of, even if not planning to use a plate reader. The framed filters used with all our 96-well systems simplify filter handling. Our 48-well reusable chamber (stock # AP48) is also widely used and frequently cited in the literature.
  • To quantify by cytometer, we recommend either our disposable ChemoTx® 96-well system or one of our MB-series 96-well reusable chambers, so you may use a microplate centrifuge to facilitate cell removal from the bottom of the filter.

Which filters should I use?

What pore size should I use?

Start with a pore size larger than the nuclei, but not so large that the cells can easily drop through the pores. Other factors including pore density may also enter into the selection of a pore size. Pore density generally decreases as pore size increases. Please see Experiment Design for more information on how this may affect your choice.

What filter type should I use?

  • We recommend PVP-free polycarbonate track-etch (PCTE) filters for most migration assays. Polyvinylpyrollidone (PVP) is a wetting agent that is not generally considered necessary with the pore sizes used for cell migration. See PVP treatment for more information.
  • If you plan to follow a protocol using cellulosic material, we recommend our 48-well chambers (stock# AP48 or AC48 with filter retaining hardware), or our 12-well (stock # AA12) or 10-well (stock # AA10) chambers. Please see Staining Cellulose Nitrate Filters and consult the literature before choosing this option.

What Neuro Probe accessories should I buy?

  1. When using 25x80mm polycarbonate filters, we recommend purchasing an accessory pack (stock # P48AP). This provides the tools you need to manipulate the filter and remove non-migrated cells. Purchasing an extra pair of filter clamps (1 each of stock # P48CL and P48CS) will allow you to use one pair when staining and maintain a clean pair.
  2. All of the filters for our 96-well chambers are bonded to rigid frames. Additional Neuro Probe accessories are generally not necessary. Some labs find it convenient to use our filter wiper (stock # P48W) to remove non-migrated cells.
  3. For cellulose nitrate filters, we recommend ordering one or 2 sets of filter clips (stock # SSC4) for use during staining, as well as to attach the filter to a slide for viewing.

Do I need coated filters for a migration assay?

Not in most cases, but some cell types perform more consistently when used with a coated filter. For example, endothelial and smooth muscle cells are typically used with filters coated with collagen, fibronectin, or a similar extra-cellular matrix protein. Neuronal cells may require filters coated with poly-D-lysine or poly-L-lysine. We recommend a search of the literature for more information.

How can I coat the filters?

1. For the ChemoTx® system, pipette your protein solution onto the well sites, making sure that the entire site inside each ring is coated. This usually requires 8-10µL for 101-series or 111-series and 20-25µL for 106-series or 116-series filters. To obtain a more consistent coating thickness, carefully remove fluid as necessary. Allow the coating to dry before using or coating the second side. Warning: Do not coat over the hydrophobic rings. This will compromise their performance. Do not puncture the filter.

2. Coating with fibronectin:

  1. Coat PVP free polycarbonate membranes with 40µL fibronectin (2mg/mL) mixed with 10mL PBS in a petri dish.
  2. Refrigerate overnight.
  3. Float membrane over PBS to remove excess fibronectin.
  4. Hang up to dry before use.

If your membranes are not PVP free, they should be pre-washed in acetic acid to remove most of the PVP and allow the protein to adhere better to the membrane.

Similar steps would be appropriate for collagen, gelatin, etc. Note: this technique is not recommended for ChemoTx® system filters.

3. Coating with Matrigel:

  • Place a PVP free membrane on a flat surface, such as a piece of glass.
  • Tape down the full length of the long edges, using a single piece of tape that will not loosen or expand when wet. ‘Invisible’ office tape usually works well. Do not tape over any part of the membrane that will cover the wells and block migration.
  • Place a small amount of Matrigel near the center of the membrane.
  • Lay a glass stirring rod, or similar smooth object, across the tape and spread the Matrigel until you have a smooth, even coating that will be approximately the thickness of the tape.
  • Remove the tape carefully, or cut the tape along the edge of the membrane. Do not tear the membrane.

Modify these steps as necessary for framed filters or 13mm diameter filters. If your membranes are PVP treated, washing them with acetic acid will remove most of the PVP so the coating adheres better. Note: this technique is not recommended for ChemoTx® system filters.

How should I place the filter in my chamber?

There are 2 visibly different sides to the filter material, one shiny and the other dull (or matte). The orientation should not affect the migration. We recommend that you select one side and use it consistently.

How many cells should I load? At what concentration?

  • Our general suggestion is that you initially load 1 to 2 cells per pore. The number of pores per site is calculated by multiplying the exposed filter area by the pore density. Data for this calculation can be found on our Experiment Design page.
  • Desired number of cells * 1000/well volume (in µL) = the required cells/mL.

What if I am seeing no migrated cells?

The most frequent causes of zero migration are:

  • The cells are either dead or inactive. Contamination of the gasket is one possible cause of cell death. Replacement Gaskets are available for our chambers. Many researchers keep a spare gasket on hand.
  • Either the chamber is not assembled properly, or the lower wells are not filled sufficiently, so the fluid in the lower wells does not contact the bottom surface of the filter.
  • The pore size is too small to allow migration of the cells. We offer filters in a wide range of pore sizes to optimize your assay.
  • The blue separator paper (that comes interspersed with the filters) has been placed in the chamber rather than the (translucent) polycarbonate filter.

When you have active cells and an appropriate filter contacting the fluid in the lower wells, there should be at least some random migration. Inactive chemokines may result in little or no differential between test sites and negative controls, but will not normally result in zero migration.

Will I get crosstalk in my fluorescence reader using your clear microplates?

  • If your reader excites and reads one well at a time, any effects of crosstalk should be minimal.
  • If your reader excites and reads multiple wells at once, we recommend first testing our plates with a serial dilution of labeled cells to determine if the sample data is acceptable. If you choose to use a different plate, our 96-well funnel plate (stock # FP1) will allow the transfer of samples.

What is a “Boyden Chamber”?

The term “Boyden chamber” is used generically in the literature for any instrument used to study trans-membrane chemotaxis as originally studied by Dr. Steven Boyden in the 1960’s. It is also referred to as “trans-well migration.” By this definition all Neuro Probe migration chambers are Boyden chambers.