ChemoTx® system instruments include specially enhanced framed filters. Please see special handling instructions at framed filters.
A general discussion of cell-activity experiments using ChemoTx® instruments is available at cell-activity assays.
- Preparing the ChemoTx® Instrument and Filling the Microplate Wells
- Adding Cell Suspension
- Reading the ChemoTx® Filter and Microplate
- Suggested Reading
Preparing the ChemoTx® Instrument and Filling the Microplate Wells
|1. Working on a clean flat surface, peel the cover off the container as indicated on the label. Using the thumb and finger cutouts on the long sides and touching only the edges, lift the clear plastic lid and microplate out of the container. Leave the framed filter in the container.|
|2. Place the microplate on the surface well-side up; the lettering should be right reading, with the well labeled A1 in the upper left corner. Do not touch the wells.|
|3. Set your pipette to dispense 29µL if your ChemoTx microplate has 30µL wells (set to 299µL if using 300µL wells). The ChemoTx system is designed to allow for variations in delivered volume of ± 2µL (± 4µL for 300µL wells) with good results. Please see pipette calibration if you are not already familiar with the process of filling a ChemoTx microplate.|
|4. Using the pipette, fill the microplate wells with test solutions such as positive and negative controls and unknowns|
|5. Touching only the edges of the frame, take the framed filter from the container and position it over the filled microplate printed side up, with the A1 corner at the upper left. Set the frame down on the microplate so that the four pins in the microplate enter the corresponding holes in the filter frame. Press the frame down firmly at each corner until the frame makes contact with the perimeter of the microplate.|
|6. Examine the sites to be sure the fluid makes contact with the filter at each completely filled site. For an explanation of how the wells and filter sites are designed and how they should look at this stage, please see pipette calibration.|
In addition to normal negative and positive control sites, we recommend using a set of calibration sites. (See controls for more about these and other kinds of control sites.)
Pipette into one row or column of wells a known serial dilution of the cell suspension to be used. For example, if 10,000 cells per site are used in the experiment, pipette 10,000 cells into well A1, 5,000 cells into well B1, 2,500 into well C1, …, 78 cells into well H1.
It is most convenient to pipette the same volume into these wells as is used on the sites on top of the filter. (If you are using a filter with a 5.65mm site diameter on a microplate with 30µL wells, you will have to adjust the volume of fluid in this technique.)
Do not pipette cell suspension on the filter sites above these calibration wells.
When the plate is read, this serial dilution serves as a standard or metric with which to compare the readings from the experimental wells.
Adding Cell Suspension
The number of cells per milliliter in the cell suspension can be calculated, provided the optimum density of cells (cells per mm2) on the top side of the filter is known. The exposed filter area of the site in mm2 multiplied by the cells per mm2 will give the optimum number of cells per site, and that number along with the volume of cell suspension to be placed at each site will give the number of cells per milliliter of media in the suspension.
The optimum density of cells on the filter must be determined as an integral part of the design of the cell-activity assay. Please see cell-activity assays for a discussion of this process.
- Pipette a drop of cell suspension onto each site on the filter top, except over any serial-dilution wells prepared as recommended above. If the sites are 3.2mm in diameter (with 8mm2 of exposed filter area), put 20µL to 25µL of cell suspension on each site. If the sites are 5.65mm in diameter (with 25mm2 of exposed filter area), put 50µL to 60µL of cell suspension on each site.
- Hold the pipette vertical and express the cell suspension slowly and evenly. We recommend a pipette that has a slow, smooth action; electronic multi-channel pipettes are ideal for this application. A hydrophobic mask on the filter’s top side causes each drop of cell suspension to stay within the perimeter of its site on the filter; no top plate with top wells is needed.
- Cover the microplate and filter with the clear plastic lid and incubate. For most chemotaxis assays the filled instrument is incubated at 37ºC in humidified air with 5% CO2. Incubation time depends on the type of cells and the experimental fluids being used. See incubation time for methods of determining the optimum time for your assay.
Reading the ChemoTx® Filter and Microplate
After incubation cells can be counted on the filter, in the microplate, or both.
ChemoTx® framed filters and microplates are designed to be counted in automated reading equipment. Please see automated readers and microplate specifications for dimensions.
See also: Pipette Calibration, Experiment Design, and Setting up Controls.
Bozarth, Penno, and Mousa. “An Improved Method for the Quantitation of Cellular Migration: Role of Integrin in Endothelial and Smooth Muscle Cell Migration.” 1997, Methods in Cell Science, 19, 179-187.
Frevert, Wong, Goodman, Goodwin, and Martin. “Rapid Fluorescence-based Measurement of Neutrophil Migration in Vitro.” 1998, Journal of Immunological Methods, 213, 41-52.
Penno, Hart, Mousa, and Bozarth. “Rapid and Quantitative in Vitro Measurement of Cellular Chemotaxis and Invasion.” 1997, Methods in Cell Science, 19, 189-195.
O’Leary and Zuckerman. “Glucocorticoid-mediated Inhibition of Neutrophil Migration in an Endotoxin-induced Rat Pulmonary Inflammation Model Occurs Without and Effect on Airways MIP-2 Levels.” 1997, American Journal of Respiratory Cell and Molecular Biology, 16, 267-274.
ChemoTx® is a registered trademark of Neuro Probe, Inc.