Nexcelom Bioscience

978-327-5340

Perform and Quantify Transwell-Based Chemotaxis using Celigo

  1. Perform, image and quantify chemotaxis assays in microwell plates
  2. Automatic direct cell count and quantification of migrated cells
  3. Acquire Hi-res bright field and fluorescent images of migrated cells

Perform and Quantify Transwell-Based Chemotaxis by Direct Cell Count

transwell illustration

Suspension cells located inside the insert migrate through the porous membrane toward the chemoattractant from the bottom plate. Celigo is used to automatically count migrated cells in the bottom plate.

Imaging and Quantification of MCP-Mediated Chemotaxis Assay

Protocol

  1. Seed cells the transwell insert overnight
  2. Add regular cell media to the bottom of the wells
  3. At various concentrations add MCP (monocyte chemoattractant protein) to the bottom of the wells and incubate for 4 hours
  4. After four hours remove the transwell insert and image the migrated THP-1 cells

Images of dose dependent MCP-mediated chemotaxis

Images of dose dependent MCP-mediated chemotaxis

Bright field images at each monocyte chemoattractant proteinĀ (MCP) dose were acquired and automatically quantified using the Celigo image cytometer.

Blue outlines indicate directly counted cells that migrated through the transwell insert into the bottom of the well.

Direct cell counts of migrated THP-1 cells

Direct cell counts of migrated THP-1 cells

Plate based results are automatically displayed with a thumb nail of the image and the number of counted cells for each analyzed well.

Dose response migration results

Dose response migration results

Data on a per-well basis is exported to excel as a .csv file for further analysis.

From the graphed data above, we observed that the highest number of migrated THP-1 cells occurred at 1 nM MCP concentration.

Determination of viability of MCP- treated THP-1 cells

Determination of viability of MCP- treated THP-1 cells

To determine whether MCP at various concentration had a negative effect on THP-1 cells, the cells where stained with propidium iodide (PI) and imaged.

Above are representative bright field and fluorescent images show very few PI positive dead cells.

Viability data of MCP-treated THP-1 cells

Viability data of MCP-treated THP-1 cells

Based on the Celigo analyzed data we can conclude that MCP did not have any negative effects on THP-1 cells at any concentration.

The acquired averages and standard deviations across all samples showed minimal variation.

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