Chemotaxis Assay Using Microchemotaxis Chambers (Modified Boyden Chamber)
互联网
1238
Overview
Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Filter Preparation (see Hint #2 )1. This procedure uses 25 x 80 mm polycarbonate filters, polyvinylpropylene (PVP) free, pore size 3, 5, 8 or 10 μm (Poretics or Neuro Probes) (see Hint #3 ). If using leukocytes, skip to Section B.
2. Prepare 3 to 5 ml of Rat Tail Collagen Solution.
3. Pour the Rat Tail Collagen Solution into a shallow vessel and submerge the filters in this solution with forceps (see Hint #4 and Hint #5 ).
4. Incubate for 2 hr at 37°C.
5. Remove the filter from the solution with forceps and allow to dry on a Kimwipe.
B. Cell Preparation
1. Grow adherent cells to 80% confluence. For cells grown in suspension, skip to step 6.
2. Wash cells once with 2 to 3 ml per 10 cm dish of Trypsin/EDTA Solution. Aspirate solution immediately.
3. Add 2 ml of Trypsin/EDTA Solution per 10 cm dish. Incubate for 2 minutes at room temperature.
4. Add 1 ml of Cell Dissociation Buffer. Incubate for 2 minutes at room temperature.
5. Add 6 ml of Serum Containing Medium.
6. Triturate the cells vigorously until a single-cell suspension is achieved.
7. Count the cells (see Protocol ID#1934 ).
8. Pellet the cells by centrifugation at 1000 rpm in a table-top centrifuge (800-1,000 X g) for 5 minutes. Aspirate the medium and resuspend the cell pellet in 20 ml of Chemotaxis Buffer.
9. Pellet the cells as in step 8. Aspirate the medium and resuspend the cells in Chemotaxis Buffer at 0.5 to 1 X 106 cells/ml. Maintain cells at 37°C until the chemotaxis chambers are ready to use.
C. Preparation of Agonist Solution(s)
1. Dilute the agonists (chemokines or other chemoattractants) to the desired concentrations in at least 200 μl Chemotaxis Buffer (see Hint #6 ). Remember to include a negative control - Chemotaxis Buffer with no chemoattractant.
2. Degas the solution under vacuum for 10 minutes to minimize the formation of air bubbles underneath the filter during the experiment.
3. Equilibrate the agonist dilutions to 37°C.
D. Assay Set Up
1. The assay is performed with a 12 or 48 multi-well microchemotaxis chamber from Neuro Probe (cat. no. AP48). In addition to the steps described below, a detailed manual is provided with the chamber.
2. Wash the chambers extensively with water and dry under compressed air.
3. The bottom portion of the chamber contains wells. Fill each well with 28 μl of a chemoattractant dilution. Ensure that bubbles are not present and that the meniscus is above the well.
4. Lower the filter (shiny side facing up) onto the wells. To do this, hold the two ends of the filter with forceps and carefully lower it until the middle portion of the filter touches the liquid in the wells, then drop the ends. Once you have dropped the filter, do not move it around.
5. Carefully place the gasket on top of the filter and then the top portion of the chamber apparatus.
6. Secure the apparatus with the screws provided. Do not tighten excessively.
7. Add 50 μl of the cells suspended in Chemotaxis Buffer to each well on the upper chamber. To avoid creating bubbles, touch the pipette tip to the side of the chamber (don't touch the filter) and expel the cells all at once.
8. Place the chamber on a level surface in a 37°C, 5% CO2 incubator. Incubate for 30 minutes to 3 hours (see Hint #7 ).
E. Fixing, Staining and Counting of Migrated Cells
1. Hold the assembly together with your hands and remove the screws. Turn the entire assembly upside down, and pull the cell source chamber (now on the bottom) and gasket away from the rest of the assembly. The filter should be stuck to the gasket. The side now facing up contains the migrated cells.
2. Grab the edges of the filter with forceps and lift it off of the gasket. Wash the upper side (now facing down) of the filter to remove excess cells as described in the manual from Neuro Probe.
3. All solutions for fixing and staining the cells are provided in the Diff-Quick Stain Set (Fisher Scientific). Use forceps to transfer the filter between solutions (see Hint #4 ).
4. Submerge the entire filter in Fix Solution for 4 minutes.
5. Transfer the filter to Solution #1 and incubate for 5 minutes.
6. Transfer the filter to Solution #2 and incubate for 5 minutes.
7. Transfer the filter to ddH2 O to remove excess stain.
8. Transfer the filter onto a glass slide with the migrated cells facing up; allow it to dry.
9. Use a microscope set at 200x magnification to count the number of stained cells in 3 to 5 fields. For each agonist condition, a migration index is calculated by dividing the number of cells that migrated in response to the agonist by the number of cells that migrated in response to Chemotaxis Buffer alone (see Hint #8 ).
Solutions
Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
| RPMI 1640 medium (Gibco) | ||
| Diff-Quick Stain Set (Fisher Scientific) | ||
| Chemotaxis Buffer |
25 mM Hepes, pH 7.4 1% (v/w) Endotoxin-free Bovine Serum Albumin prepare in RPMI 1640 medium |
|
| Rat Tail Collagen Solution (see Hint #1) |
50 μg/ml rat tail collagen, Type 1 (see Protocol ID#1960 ) 0.2 M Acetic Acid prepare in RPMI 1640 medium |
|
| Serum Containing Medium |
10% (v/v) serum (such as fetal bovine serum) in RPMI 1640 medium |
|
| Cell Dissociation Buffer (GIBCO) |
Mg2+ and Ca2+ free |
|
| Trypsin/EDTA Solution (GIBCO) |
0.5 g/liter Trypsin 0.2 g/liter EDTA |
|
Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Cell Dissociation BufferTrypsin
Endotoxin-free Bovine Serum Albumin
RPMI 1640
Rat Tail Collagen
EDTA
Acetic Acid
Fetal Bovine Serum
HEPES
Protocol Hints
Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Collagen should be stored under acidic conditions; it is insoluble at neutral pH.2. When using immune cells such as neutrophils, do not coat the filter. This protocol can be used to study the migration of adherent cells, such as HEK-293 cells. Coating the filters with Rat Tail Collagen was found to be important for this cell line. For other cell lines, determine empirically whether coating is necessary.
3. The pore size of the filter depends on the type of cell being used. For Human neutrophils, use filters with 5 μm pores; for Mouse neutrophils, use filters with 8 μm pores. HEK-293 cells use filters with 10 μm pores.
4. Never touch the filters with bare hands.
5. The filters have a tendency to float on the solution and dry out during the coating procedure. To avoid this, rock the solution every 10-15 minutes.
6. Chemokines are generally used in a concentration range from 0.01 nM to 100 nM. However, the optimal concentration range for a specific chemokine or chemotactic factor varies. The literature should be consulted for the appropriate concentration range for a factor of interest.
7. The incubation time depends on the type of cell being used. Immune cells such as neutrophils require shorter incubation times (30 minutes), while adherent cultured cells such as HEK-293 cells require longer incubation time (3 hours).
8. A checker-board analysis distinguishes chemotaxis (the directed migration of cells in response to a gradient of chemotactic factor) from chemokinesis (increase in the speed or frequency of movement induced by a factor). This can be achieved with the same apparatus and procedure outlined here except that migration is measured under non-gradient conditions, (i.e., with an equal concentration of chemoattractant in the upper and lower chambers). This assay variation was originally described by Zigmond et al. (see Citation #1 ).
1. Zigmond, S. H., and Hirsch, J. G. Leukocyte locomotion and chemotaxis. New methods for evaluation, and demonstration of a cell-derived chemotactic factor J. Exp. Med 1973; 137: 387-410.
