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IncuCyte™ Protocols : Chemotaxis Invasion


General IncuCyte™ Chemotaxis Cell Invasion Protocol

General IncuCyte™ Chemotaxis Cell Invasion Protocol

This protocol provides an overview of the IncuCyte™ Chemotaxis Cell Invasion Assay methodology.

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IncuCyte™ Chemotaxis Cell Invasion General Protocol

This protocol provides an overview of the IncuCyte™ Chemotaxis Cell Invasion Assay methodology and is compatible with the IncuCyte ZOOM® instrument. Measurements of chemotactic cell invasion can be made using nuclear labeled or unlabeled cells, however, we recommend labeling cells with a live-cell nuclear label (e.g. IncuCyte® NucLight™ reagents) to ensure optimal performance.

general protocol

General Protocol

1. Pre-cool a ClearView Chemotaxis plate in a CoolBox system containing a frozen cold pack and CoolSink plate (4°C), for 5 minutes. Learn How to set up CoolBox™ accessories for IncuCyte™

Note: It is important to keep close temperature control of biomatrix materials such as Collagen-1 and basement membrane extract (BME) to prevent unwanted gelling. The IncuCyte™ Cell Invasion Kit (Cat. No. 4444) includes a specialized CoolBox system to ensure the temperature of your assay plate and biomatrix materials are maintained between 4-8°C – preventing premature polymerization and eliminating edge effects. Crushed ice can be used as an alternative however non-uniform cooling can lead to assay variability.

2.Add 150 μL of D-PBS (4°C) to each well of the pre-chilled ClearView reservoir plate. Replace the ClearView insert and allow the membrane to prime for 20 minutes within the CoolBox.

How to prime the ClearView membrane

3.While the ClearView plate is priming, prepare your biomatrix reagent at the desired working concentration as per the manufacturer's instructions.

Note: Keep biomatrix reagents cool (4-8°C) by placing them in a CoolBox system or by embedding them in ice. Biomatrix solutions can be viscous. Prepare a large dead volume to ensure available biomatrix for transfer to the assay plate (e.g., prepare 4 mL biomatrix solution to provide 20 μL per insert well).

The IncuCyte™ Chemotaxis Cell Invasion assay is compatible with your choice of biomatrix gel. Recommended biomatrix proteins include: Cultrex® 3-D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract (Trevigen 3445-005-01) and Cultrex® Rat Collagen I (Trevigen 3440-100-01).

The required biomatrix density will be dependent on the matrix and cell types used. For HT-1080 cells we recommend BME (5 mg/mL) diluted in assay medium or Collagen I (1 mg/mL) diluted in neutralizing buffer (DMEM, Sigma D2429, + 7.5 g/L sodium bicarbonate + 0.004 g/L folic acid + 1% GlutaMax). When preparing collagen I it is important that it is properly neutralized to ensure cell health is maintained and gelling is uniform.

4.Harvest your cells and resuspend the pellet in the biomatrix solution. Cell density will need to be optimized for each cell type used; however, we have found that 1,000 cells per well is a reasonable starting point.

How to resuspend your cells within a biomatrix

Calculation: 50,000 cells/mL x 0.02 mL/well = 1,000 cells/well

Note: Some cell types may require reduced exposure to Fetal Bovine Serum (FBS) before initiating the transmembrane invasion assay (e.g., HT-1080s starved in F12 + Insulin-Transferrin-Selenium for ~20 hours).

5.Seed cells (20 µL per well, 1,000 cells per well) into every well of the cooled ClearView insert plate.

How to add a biomatrix cell suspension to the ClearView insert

Note: Use a pre-cooled reservoir boat when seeding cells.

6.Centrifuge the ClearView plate in a cooled centrifuge (set at 4°C) for three minutes at 50 x g.

7.Place the ClearView plate at 37°C on a pre-warmed CoolSink and allow the biomatrix to polymerize for 30-60 minutes.

How to polymerize your biomatrix

8.Gently add 40 µL of assay medium ± modulators of invasion on top of the biomatrix:cell layer in the insert wells.

Note: If adding modulators of invasion, the working concentration should be 1.5x the desired final concentration to account for the volume of the biomatrix:cell layer.

9.Add 200 μL of desired chemoattractant, or control, to the appropriate wells of a second reservoir plate. Carefully transfer the insert into the pre-loaded reservoir plate. Learn how to transfer the insert. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate.

10.Place the ClearView cell migration plate into the IncuCyte ZOOM® instrument and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, wipe away any condensation that remains on the outside of the plate lid or bottom of the reservoir.

11.Image the plate in the IncuCyte ZOOM® instrument with a 10x objective, using the Chemotaxis Scan Type with “Phase” and the appropriate "Red" or "Green" channels selected dependent on the fluorescent label used.


Best Practices for Avoiding Bubbles

Air bubbles trapped on either side of the membrane can interfere with proper focusing and image processing. We recommend the following techniques to eliminate bubbles from your experiment:

Reverse pipette during the biomatrix:cell addition step and when adding medium ± treatment on top of the polymerized biomatrix:cell layer. Reverse pipetting reduces the risk of splashing or bubble formation. In reverse pipetting, the volume aspirated into the tip is larger than the volume delivered to the receiving vessel:

1.Press the plunger to the second stop.

2.Dip the pipette-tip into the solution.

3.Release the plunger until the starting position has been reached.

4.Move the pipette-tip to the receiving vessel.

5.Dispense the liquid by pressing the plunger to the first stop. SOME LIQUID WILL REMAIN IN THE TIP.

6.Repeat steps 2–5 throughout the plate.

Remove bubbles at the liquid surface by gently squeezing a wash bottle (containing 100% ethanol with the inner straw removed) to blow vapor over the surface of each well.

When replacing the ClearView insert into the pre-filled reservoir do so at a slight angle to ensure the reservoir liquid displaces all air trapped under the membrane.

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Detailed Chemotaxis Cell Invasion Protocol

Detailed Chemotaxis Cell Invasion Protocol

A detailed demonstration protocol designed to enable you to run a successful IncuCyte™ Chemotaxis Cell Invasion Assay with nuclear labeled HT-1080 fibrosarcoma cells.

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IncuCyte™ Chemotaxis Cell Invasion General Protocol

The following protocol is a detailed example designed to enable you to run a successful transmembrane cell invasion assay using the IncuCyte™ Chemotaxis Cell Invasion Assay. Note that the protocol does not include a description of any experiments required for optimization. Here we specifically describe the use of the IncuCyte ZOOM® instrument for establishing and quantifying chemotactic invasion of HT-1080 cells.


Materials

NucLight™ Red HT-1080 (Essen BioScience 4485) cells in culture (T75, 37°C, 5% CO2) in:

Growth medium: F12 (Invitrogen 11765) + 10% FBS + 1% Glutamax + 1% Pen/Strep. + 0.5 μg/ml Puromycin

IncuCyte™ ClearView 96-Well Cell Migration Plate (Essen BioScience 4582 or 4599)

2x IncuCyte™ ClearView Cell Migration Reservoir Plate (Essen BioScience 4600 or 4601)

0.25 % Trypsin/EDTA (Life Technologies 25200)

D-PBS (w/o Ca2+, Mg2+, Life Technologies 10010)

Insulin-Transferrin-Selenium (Life Technologies 41400-045)

Fetal Bovine Serum (Sigma-Aldrich F2442-500 mL)

Cultrex® 3D Culture Matrix™ Reduced Growth Factor Basement Membrane Extract (Trevigen 3445-005-01)

IncuCyte™ Chemotaxis Cell Invasion Accessories (Essen BioScience 4444)

2x CoolBox 96F System configured with CoolSink 96F (Essen Bioscience1500-0078-A00)

CoolBox 30 System configured with CoolRack M30 (Essen Bioscience 1500-0079-A00)

CoolSink 96F for 96-well flat bottom plates (Essen Bioscience 1500-0080-A00)


Protocol

Day 0

1)Thaw Basement Membrane Extract (BME) on ice or in a pre-chilled CoolBox. Once thawed keep at on ice until ready to use.

NOTE: Culture should be below 80% confluence. A T-25 flask at 75–80% confluence generally yields enough cells to perform an entire 96-well

2)ChemotaxisCell Migration or Invasion Assay. Replace with serum-free assay medium, F12 + 1x Insulin-Transferrin-Selenium (ITS). Return culture to incubator for 20 hours to serum starve the cells.

Day 1
PREPARE PLATE

1)Thaw Basement Membrane Extract (BME) on ice or in a pre-chilled CoolBox. Once thawed keep at on ice until ready to use.

Note: BME will gel at temperatures above 8°C. It is extremely important to keep this reagent at 4°C to prevent polymerization during assay set up.

1)Pre-cool a ClearView Chemotaxis plate in a CoolBox system containing a frozen cold pack and CoolSink plate (4°C), for 5 minutes.

Learn How to set up CoolBox™ accessories for IncuCyte™

Note: It is important to keep close temperature control of the ClearView Chemotaxis plate. The IncuCyte™ Cell Invasion Kit (Cat. No. 4444) includes a specialized CoolBox system to ensure the temperature of your assay plate and biomatrix materials are maintained between 4-8°C – preventing premature polymerization and eliminating edge effects. Crushed ice can be used as an alternative however non-uniform cooling can lead to assay variability.

2)Add 150 µL of D-PBS (4°C) to each well of the pre-chilled ClearView reservoir plate. Replace the ClearView insert and allow the membrane to prime for 20 minutes within the CoolBox.

How to prime the ClearView membrane

3)While the ClearView plate is priming, dilute the BME to 5 mg/mL using chilled assay medium.

Note: This reagent will be used at a volume of 20 µL per insert well, but prepare the volume you need with excess (e.g., prepare 4 mL biomatrix solution to provide 20 µL per insert well).

PREPARE CELLS

4)Remove medium from culture and gently rinse with D-PBS. Harvest cells and perform a cell count (e.g., trypan blue staining + hemacytometer).

5)Transfer 200,000 cells to a 15 mL conical tube and centrifuge at 1000 RPM for 4 minutes.

6)Resuspend the cell pellet in 4 mL of BME (5mg/mL) (prepared in step 4). Final cell stock will be 50,000 cells/mL. Keep on ice at all times.

How to resuspend your cells within a biomatrix

SET-UP PLATE

7)Using a multi-channel pipette and reverse pipetting technique, seed cells (20 µL per well, 1000 cells per well) into every well of the ClearView insert plate in the CoolBox (4 °C).

How to add a biomatrix cell suspension to the ClearView insert

Note: Use a pre-cooled reservoir boat when seeding cells.

8)Centrifuge ClearView plate in a cooled centrifuge (set at 4°C) for three minutes at 50 x g.

9)Place the ClearView plate at 37°C on a pre-warmed CoolSink and allow the BME to polymerize for 30-60 minutes.

How to polymerize your biomatrix

10)During incubation, prepare chemoattractants (e.g. 2-fold serial dilutions of 10% FBS in F12 medium + ITS). Assay medium alone (F12 + ITS) should be used as a control.

11)Using a manual multi-channel pipette, add 200 μL of the chemoattractants and control assay medium to the appropriate wells of the second reservoir plate.

12)Using a manual multi-channel pipette, gently add 40 µL of assay medium to all insert wells on top of the BME:cell layer.

13)Carefully transfer the insert into the pre-loaded reservoir plate. Be careful not to introduce bubbles which can become trapped below the membrane when placing the insert into the pre-filled reservoir plate.

Learn how to transfer the insert

INITIATE SCANNING

14)Place the ClearView cell migration plate into the IncuCyte ZOOM® instrument and allow the plate to warm to 37°C for at least 15 minutes. After 15 minutes, wipe away any condensation that remains on the outside of the plate lid or bottom of the reservoir

15)In the IncuCyte ZOOM® software, schedule 24 hour repeat scanning (10x) every 2-3 hours. Typical assay duration is 72 hours.

1.Objective: Ensure 10x objective is installed

2.Vessel Type: Select “ClearView Cell Migration”

3.Channel Selection: Select “Phase” + “Red” (800 ms acquisition time)

4.Scan Mode: Select “Chemotaxis (Top/Bot)” scan type and desired Scan Pattern

5.Note: The IncuCyte™ instrument estimates a scan time of 33 min per plate (phase and red); however, the actual scan time can take longer.

Note: We recommend plotting bottom side fluorescent nuclear count as the assay metric


Data

Determination of NucLight Red HT-1080 invasive and migratory response toward FBS

Figure 1. Determination of NucLight Red HT-1080 invasive and migratory response toward FBS. Plate map and corresponding microplate graph for FBS agonist curves. Serum starved NucLight Red HT-1080 cells were either embedded in 5mg/mL BME (invasion) or seeded directly onto the ClearView Cell migration plate membrane (migration control) at 1,000 cells per insert well. The indicated concentration of FBS was added to the reservoir plate and data were collected for 96 hours at 2 hour intervals. Each well is individually graphed illustrating the difference in kinetic movement of migrating and invading cells.

Representative morphological images of f HT-1080 cells

Figure 2. Representative morphological images of f HT-1080 cells. Migration: Images A, B, and C show the time-lapse progression of HT-1080s cells migrating toward 10% FBS. HT 1080 cells have a flat appearance and migration to the bottom side of the membrane (cells that are out of focus) can be seen prior to 24 hrs. Invasion Images D, E, and F show HT-1080 cells invading through the BME (5 mg/mL) toward the membrane pores. Cells have an elongated morphology, sending out laemellipodia or extensions to drive movement through a dense gel layer.

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