Category by field: Cancer Biology > Metastasis

Category by field: Cell Biology > Cell analysis

Category by organism: Mammalia > Cell line > Cell biology

Chemotaxis Assays

Author names(Please put authors’names in an order of first name, last name).

Corresponding author: name, email and affiliation

[Abstract]Cell migration is a highly integrated, multi-step process that plays an important role in the progression of various diseases including cancer. Specifically, migration of endothelial cells (ECs) is a key step in the growth and development of new blood vessels. Here, we describe the directional cell motion along a concentration gradient of a chemical, also termed‘chemotaxis’. The most widely used assay for measuring chemotaxis is the modified Boyden chamber assay. The Boyden chamber consists of two compartments separated by a polycarbonate microporous filter with a defined pore size. Cells are loaded into the upper compartment and allowed to migrate through the pores into the lower compartment, in which the chemotactic agentis loaded. After an appropriate incubation time, the membrane is fixed and stained, and the cells that have migrated to the lower side arecounted.

Materials and Reagents

  1. Bone marrow primary endothelial cells (ECs) from multiple myeloma patients
  2. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM)(Euroclone, catalog number: ECM0103L)
  3. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number:F9665)
  4. PBS without Ca2+ and Mg2+ (Euroclone, catalog number:ECB4004L)
  5. Trypsin/EDTA (Euroclone, catalog number: ECB4004L)
  6. Recombinant human Vascular Endothelial Growth Factor (VEGF) (10ng/ml; Sigma Chemical Co., catalog number:SRP4363)
  7. Recombinant human basic Fibroblast Growth Factor 2 (FGF2) (10ng/ml; Peprotech Inc., catalog number: 100-18B)
  8. Polycarbonate membrane 8 μm pore-size (Neuro Probe, catalog number:417-0014)
  9. Fibronectin (10μg/ml; Sigma-Aldrich, catalog number:F0895)
  10. Diff-Quick Stain Kit (Labex AB, catalog number: BBS28K)
  11. Methanol
  12. Tris 0.05 M pH 7.5 buffer(See Recipes)

Equipment

  1. 48-well micro chemotaxis chamber, including: a lower chamber, a silicone gasket, an upper chamber and screws (Neuro Probe, catalog number: AP48)
  2. Glasstic slide with grids (Sentinel Diagnostic, catalog number:22-270141)
  3. Wiper blade
  4. Centrifuge(Note: For the unit of spinning speed, rpm can only be used when its centrifuge model number is known otherwise please use RCF (x g) instead.)
  5. 37°C 5% CO2 Cell culture incubator
  6. Microscope
  7. Filter clamp
  8. Cotton swap

Procedure

  1. Polycarbonate membrane 8-μm pore size is coated with 10 μg/ml fibronectin in 0.05M Tris pH 7.5 coating buffer.

Note: XXXXXXXXXXXX (If applicable; this is for extra notes and tips for this step)

  1. The membrane is incubated in the 5% CO2 incubator at 37°C overnight.
  2. Bone marrow primary ECs are washed twice with PBS, trypsinized and counted using Glass slide with grids.
  3. The cells are pelleted at 1,500 rpm for 5 minutes.
  4. The supernatant is removed and 1X106 cells/ml resuspended in serum-free DMEM.
  5. The bottom plate of the chemotaxis chamber is oriented on a flat surface so that the NP trademark is at the upper left.
  6. 28μl of DMEM with 1.5% FBS with or without (negative control) 10 ng/ml VEGF and 10 ng/ml FGF2 (or other chemoattractant molecules) are loaded in the bottom wells of the micro chemotaxis chamber.
  1. A corner of the membrane is cut off and oriented on the filled wells with the cut corner at the upper left, with the shiny side facing up.
  2. A silicone gasket is applied on the bottom plate, aligning its NP trademark with the trademark of the bottom plate.
  3. The top compartment of the chamber apparatus is placed on top of the silicone gasket.
  4. The apparatus is secured with the provided screws.
  5. 5X104 ECs in 50μl of serum-free DMEM are added 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).
  1. The chamber is placed on a level surface in the 37°C 5% CO2 incubator for 6 hours. The incubation time varies considerably depending on the cell type and chemotactic factor.
  2. The entire assembly is turned upside down and the screws are removed.
  3. The cell source chamber and the gasket are pulled away from the rest of the assembly.
  1. The migrated cells are now facing up on the filter.
  2. One end of the filter is caught with the large filter clamp and the non-migrated cell side wetted in a dish containing PBS. Cells on the non-migrated cell side of the filter are wiped off by drawing the filter up over the wiper blade. The wiper is cleaned with a cotton swab and this step repeated at least twice.
  3. When the filter is dried, it is immersed in methanol, then stained with Diff-Quik dye.
  4. The wet filter cell-side up is placed on a microscope slide to be dried.
  5. Then, the filter is ready for counting the number of stained cells in 3 to 5 fields at 200x magnification (Figure 1).

Figure 1. Title xxx (Bold font). Legend xxx xx xx.(if applicable)

Table 1. Title (Bold font) (if applicable)

Video 1: Title (Bold font) (if applicable; a short video (smart phone quality) for certain step would be very helpful for readers.. . Please see an example at

[Notes] (if applicable; this section is for extra notes and tips for the whole procedure)

  1. XXXXXXXXXXXXXXXXXXXXXXXXXXXXX
  2. XXXXXXXXXXXXXXXXXXXXXXXXXXXXX

Recipes

  1. Tris 0.05M pH 7.5 buffer (100ml)

Mix 0.6 g of Tris base with 80 ml dH2O,

pH to 7.5 with HCl.

Add dH2Oto 100 ml.

Filter sterilize (0.45 μm).

Store at 4°C.

References

  1. *Anwari, K., Webb, C. T., Poggio, S., Perry, A. J., Belousoff, M., Celik, N., Ramm, G., Lovering, A., Sockett, R. E., Smit, J., Jacobs-Wagner, C. and Lithgow, T. (2012). The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex. Mol Microbiol 84(5): 832-844.

* How to cite this protocol: please cite Reference 1.

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