Using Automated Readers

See also Microplate Specfications.

In a cell-activity assay, a cell line and chemical chemotactic factor are the focus of the assay, and we assume that the effects of the chemical on the cells’ activity is to be demonstrated using an instrument that includes sites at which the chemical is in contact with the under side of a membrane filter while a suspension containing the cells lies on top of the filter. (There is a general discussion at cell-activity assays.)

A number of automated readers are now in use that measure properties of cell populations in the wells of a standard format 96-well microplate. To facilitate the use of such detection systems in cell-activity assays, Neuro Probe has developed the ChemoTx® system and reusable MB-series 96-well chemotaxis chambers that use framed filters and microplates specially designed for automatic reading equipment. (To determine the suitability of various microplates for your automated reader, use the dimensional information at Microplate Specifications.)

There are four widely used types of automated detection systems: densitometric or ELISA microplate readers, fluorescence microplate readers, scintillation counters, and photoluminescence readers. There are detailed protocols for the first two at ELISA plate readers and fluorescence readers below.

With a scintillation counter, cells are treated with a radioactive tag and read in an opaque white microplate. Handling radioactive material requires appropriate precautions, and radioactive tags may influence cell function. The number of cells required at each site will vary with the tag used and the quantity of tag taken up by the cells, but typically the method offers relatively low sensitivity.

With a photoluminescence reader, cells are treated with a reagent that causes light emission and read in an opaque white microplate. The reagents are introduced after incubation, so do not influence cell migration. This method is not extensively documented in the literature, but appears to be quite sensitive

There are a variety of ways to use ELISA densitometric plate readers and fluorescence readers to count cells that have migrated across the filter membrane during incubation. Cell treatment both before and after migration varies according to the kind of cells and the type of reader being used. The counting procedures described here for the two types of readers are typical.

ELISA Plate Readers

After incubation decant or aspirate fluid from the top side of the framed filter.

If you are using cell types that firmly adhere to the filter after migrating and you wish to dislodge them, pipette a solution of two-millimolar EDTA in PBS onto the sites on top of the framed filter (into the top wells if an MB-series chamber is being used). Use 10-15µL for 3.2mm-diameter sites and about 40µL for 5.7mm-diameter sites. Incubate the chamber again for 30 minutes at 4°C; this encourages the cells to disengage from the filter. (If the cells are of a type that disengages easily from the filter, this step can be eliminated.)

Holding the filter/microplate unit by the edges only, gently wipe the non-migrated cells off the top of the filter with a cotton swab, cell harvester, or small squeegee. Hold the unit at 45 degrees over a sink or container and carefully flush the top surface of the filter with cell-suspension media or PBS. Apply the rinse to the top edge of the filter and allow it to gently flow across the filter surface. The goal is to remove non-migrated cells from the top surface without disturbing cells that have migrated through the filter. (Do not detach the filter from the microplate during these first steps.)

To move cells onto the bottom of the microplate wells from the pores and the under side of the filter, spin the microplate/filter assembly in a centrifuge at a speed and time appropriate for the kind of cells and reagents involved.

To verify cell removal, separate the filter from the microplate after centrifuging. (Use a small, thin object such as a paper clip to gently loosen the frame from the corner pins.) Stain the filter with Diff-Quik™ (available from VWR Scientific Products) or an equivalent stain, and examine the bottom surface of the filter under a microscope.

Once the migrated cells are in the microplate, any procedure that produces a color density proportional to the number of cells can be used to get a densitometric, or colorimetric, reading from an ELISA plate reader.

Typically about half the fluid in the microplate wells is aspirated out and the wells are refilled with a suitable stain; follow the protocol appropriate to that stain. (Promega’s MTT and MTS are possible stains.) To obtain a standard or metric that can be used to relate instrument readings and cell numbers, load a second microplate with solutions containing stain and known numbers of viable cells and determine optical densities proportional to the known cell numbers.

If you are using a ChemoTx instrument with 30µL wells or MP30 plates or an IB-50 or IC-25 reusable insert in an MB-series chamber, you have the option of using a 96-well funnel plate (Stock# FP1) to transfer the microplate contents to a larger clear-bottom microplate suitable for densitometric reading. Place the large openings of the funnel plate over the 30µL microplate and position the larger microplate over the funnel plate so that the small funnel openings are in the wells. Invert the unit composed of the funnel plate and two microplates and centrifuge it to move the cells into the larger microplate.

The following section illustrates the use of an ELISA plate reader in a specific case where the cells are macrophages and the dye is MTT.

Reading Macrophages Stained with MTT

After incubating the microplate/filter assembly and removing fluid from the top of the filter, incubate again with EDTA and remove nonmigrated cells from the top of the filter as described above.

Centrifuge the microplate/filter assembly between 400-500g for 10 minutes.

After centrifuging, separate the filter from the microplate and set it aside so that you can verify cell removal as described above.

Carefully aspirate about half the fluid from each well in the microplate and refill the wells with a solution of one part MTT (Sigma: 5mg/ml) to 20 parts media. Incubate the microplate for 4 hours at 37°C.

After this incubation aspirate the fluid from the wells, leaving only the crystals of stain in the bottom. Dissolve the crystals in acid isopropanol or DMSO. With acid isopropanol, the plate must be agitated to dissolve the crystals.

Read the plate in an ELISA plate reader with a 540µm filter.

Fluorescence Readers

Method I: Reading Fluorescence-Tagged Cells in the Microplate

Tag the cells with an appropriate fluorescent dye (e.g., Calcein AM from Molecular Probes). This may be done when preparing the cell suspension to be pipetted onto the top side of the filter, or it can be done after incubation, depending on the type of assay.

After incubation decant or aspirate fluid from the top side of the framed filter (from the top wells if a reusable chamber is being used).

If you are using cell types that firmly adhere to the filter after migrating, pipette a solution of two-millimolar EDTA in PBS onto the sites on top of the framed filter (into the top wells if a reusable chamber is being used). Use 10-15µL for 3.2mm-diameter sites and about 40µL for 5.7mm-diameter sites. Incubate the chamber again for 30 minutes at 4°C; this encourages the cells to disengage from the filter. (If the cells are of a type that disengages easily from the filter, this step can be eliminated.)

Holding the filter/microplate unit by the edges only, gently wipe the non-migrated cells off the top of the filter with a cotton swab, cell harvester, or small squeegee. Hold the unit at 45 degrees over a sink or container and carefully flush the top surface of the filter with cell-suspension media or PBS. Apply the rinse to the top edge of the filter and allow it to gently flow across the filter surface. The goal is to remove non-migrated cells from the top surface without disturbing cells that have migrated through the filter. (Do not detach the filter from the microplate during these first steps.)

Centrifuge the filter/microplate assembly for 10 minutes between 400-500g. This forces the cells from the pores and the under-side of the filter onto the bottom of the microplate well.

Using a small, thin object such as a paper clip, gently loosen the frame from the corner pins, detach the filter and set it aside. It can later be read in a fluorescence reader to verify that cells have been removed.

Place the microplate in a fluorescence reader to count migrated cells in the microplate wells. See Microplate Specifications to help determine how the microplate in a Neuro Probe instrument will fit your fluorescence reader.

Method II: Reading Fluorescence-Tagged Cells on the Filter

Tag the cells with an appropriate fluorescent dye (e.g., Calcein AM from Molecular Probes). This should be done when preparing the cell suspension to be pipetted onto the top side of the filter.

After incubation decant or aspirate fluid from the top side of the framed filter (from the top wells if a reusable chamber is being used).

Holding the filter/microplate unit by the edges of the frame, wipe the non-migrated cells off the top of the filter with a cotton swab, cell harvester, or small squeegee, as described above. Cell-suspension media or PBS can be used to rinse. (Do not detach the filter from the microplate during these first steps.)

Separate the filter from the microplate, and carefully wipe the top of the filter again to remove any remaining cells. Set the microplate aside.

Place the framed filter in a fluorescence reader, aligning the filter to count migrated cells on the bottom. If reading from the top, invert the filter but note that the well order will be reversed.

To count the cells that have fallen off the filter after migrating, place the microplate in the fluorescence reader and count. This technique can be used, for example, to quickly and directly calculate the percentage of migrated cells that have fallen off the filter.

 

Suggested Reading

Bozarth, Penno, and Mousa. “An Improved Method for the Quantitation of Cellular Migration” 1997, Methods in Cell Science, 19, 179-187.

PDF fileFrevert, Wong, Goodman, Goodwin, and Martin. “Rapid Fluorescence-based Measurement of Neutrophil Migration in Vitro.” 1998, Journal of Immunological Methods, 213, 41-52.

Junger, Cardoza, Liu, Hoyt, and Goodwin. “Improved Rapid Photometric Assay for Quantitative Measurement of PMN Migration.” 1993, Journal of Immunological Methods, 160, 73-79. [NOTE: The Neuro Probe chemotaxis chamber illustrated in this paper is an early prototype of the MB-series instruments; it has no hinges or spring-loaded catch.]

O’Leary and Zuckerman. “Glucocorticoid-mediated Inhibition of Neutrophil Migration in an Endotoxin-induced Rat Pulmonary Inflammation Model Occurs Without an Effect on Airways MIP-2 Levels.” 1997, American Journal of Respiratory Cell and Molecular Biology, 16, 267-274.

Penno, Hart, Mousa, and Bozarth. “Rapid and Quantitative in Vitro Measurement of Cellular Chemotaxis and Invasion.” 1997, Methods in Cell Science, 19, 189-195.

Shi, Kornovski, Savani, and Turley. “A Rapid, Multiwell Colorimetric Assay for Chemotaxis.” 1993, Journal of Immunological Methods, 164, 149-154.