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IncuCyte™ Protocols : Chemotaxis


General IncuCyte™ Chemotaxis Cell Migration Protocol

General IncuCyte™ Chemotaxis Cell Migration Protocol

This protocol provides an overview of the IncuCyte™ Chemotaxis Cell Migration Assay methodology which can be used with adherent or non-adherent cell types.

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Incucyte™ Chemtoaxis Cell Migration Assay General Protocol

This protocol provides an overview of the IncuCyte™ Chemotaxis Cell Migration Assay methodology, which can be used with adherent or non-adherent cell types. This protocol is compatible with the IncuCyte ZOOM® instrument using nuclear labeled or non-labeled cells.



general protocol

1.Seed cells of interest at 60 μL per well (40 μL per well if adding inhibitors of cell motility; refer to step 2) at an appropriate density into a ClearView 96-well insert. Typically this is done with the insert mated to an empty reservoir plate. The seeding density will need to be optimized for each cell type used, however, we have found that 1,000 cells per well for adherent cell types and 5,000 cells per well for non-adherent cell types are reasonable starting points. Learn how to seed cells

Some cell types may include reduced exposure to Fetal Bovine Serum (FBS) prior to initiating this transmembrane assay (e.g. HT-1080s starved in F12 + Insulin-Transferrin-Selenium for ~20 hours).

Some cell lines (e.g. neutrophils) may include the addition of a basement membrane extract (e.g. 50 μg/mL Matrigel® + 10% FBS) to promote light cell adherence and provide the necessary integrins for cell motility. Follow manufacturer’s recommendations for coating. Refer to Table 1 for cell line seeding density and coating recommendations. Learn how to coat

2.If pre-treating cells, prepare 3x treatments and immediately add 20 μL to the insert wells containing cells immediately after cell seeding. Triturate the cells, using a 30 μL volume, to appropriately mix the treatment, so cell exposure during pre-treatment is at 1X.

NOTE: It is important to immediately add treatment to insert wells immediately after seeding to avoid cell settling prior to treatment addition. If adherent cells attach prior to trituration, uneven cell distribution can occur.

3.Place the plate onto a level surface and allow the cells to settle at ambient temperature for 15 minutes (adherent cell types) to 60 minutes (non-adherent cell types). For adherent cell types, we recommend a continued pre-incubation with inhibitors at 37°C for 30 minutes.

4.Add 200 μL of desired chemoattractant or control to the appropriate wells of a second reservoir plate. Carefully transfer the insert ( Learn how to transfer the insert icon-video) 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 remove bubbles

5.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, remember to carefully wipe away any condensation that may have accumulated on the plate lid or bottom of the reservoir.

6.Image the plate in the IncuCyte ZOOM® instrument with a 10x objective using the Chemotaxis Scan Type.

Table 1. Validated Cell Lines and Recommendations
Table 1. Validated Cell Lines and Recommendations
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Detailed Adherent Chemotaxis Cell Migration Protocol

Detailed Adherent Chemotaxis Cell Migration Protocol

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

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Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte™ Chemotaxis Cell Migration Assay. Note, that the protocol does not include a description of any experiments included for optimizing seeding density or surface coatings. Here we specifically describe the use of IncuCyte ZOOM® instrument for establishing and quantifying the inhibition of HT-1080 serum-induced chemotaxis in the presence of Cytochalasin D.



HT-1080 Chemotaxis: Inhibition

Materials

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

F12 + 10% FBS + 1% Glutamax + 1% Pen/Strep. + 0.5 μg ml-1 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 F2442-500mL)

Cytochalasin D (Sigma-Aldrich C8273)

Protocol

Day 0

1.Remove serum-containing medium from culture and gently rinse twice with D-PBS.

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

2.Replace with serum-free F12 + 1x Insulin-Transferrin-Selenium (ITS). Return culture to incubator for 20 hours.

Day 1

1.n advance, prepare a 3x stock of Cytochalasin D (e.g. highest cell treatment of 3 μM includes a stock concentration of 9 μM) and prepare 3-fold serial dilutions.

2.Remove serum-free chemotaxis medium from culture and gently rinse with D-PBS.

3.Harvest cells and perform a cell count (e.g., trypan blue staining + hemacytometer). Centrifuge the cell suspension (1000 RPM, 4 minutes) and resuspend the cell pellet in culture medium at 25,000 cells/mL. (Calculation: 25,000 cells/mL x 0.04 mL = 1000 cells per insert well).

NOTE: When performing a chemotaxis cell migration assay without treatment, the cell seeding volume is increased to 60 μL, thus the cell seeding stock is reduced to 16,666.67 cells/mL.

4.Using a manual multi-channel pipette and reverse pipetting technique, seed cells (40 μL per well, 1,000 cells per well) into every well of the insert plate. how to seed cells

5.Add 20 μL of the prepared Cytochalasin D at 3x final assay concentration to the appropriate wells and mix by repeatedly pipetting up and down with a 30 μL volume.

6.Allow the cells to settle at ambient temperature for 15 minutes then place the ClearView Cell Migration Plate at 37°C, 5% CO2 for 30 minutes in order to pre-incubate the HT-1080 cells in the presence of Cytochalasin D.

7.Prepare F12 + ITS + 10% FBS as the chemoattractant, and F12 + ITS for the control wells.

8.Using a manual multi-channel pipette, add 200 μL of the chemoattractant and control medium to the appropriate wells of the second reservoir plate.

9.Carefully transfer the insert plate containing cells ± Cytochalasin D treatment into the reservoir plate containing medium ± chemoattractant. Learn how to transfer the insert

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.In the IncuCyte ZOOM® software; schedule 24 hour repeat scanning (10x) for every 1-2 hours.

a. Objective: Ensure 10x objective is installed.

b. Vessel Type: Select ‘ClearView Cell Migration’

c. Channel Selection: Select ‘Phase’ + ‘Red’ (800 ms acquisition time).

d. Scan Mode: Select ‘Chemotaxis (Top/Bot)’ scan type and desired Scan Pattern

e. Note IncuCyte™ estimates a scan time of 20 min per plate (phase only) and 33 min per plate (phase and red); however, the actual scan time can take longer if condensation is not properly removed.

Example of Data Generated with the Detailed Adherent Chemotaxis Cell Migration Protocol
Chemotaxis adherent protocol representative images

Representative images of NucLight Red HT-1080 cells migrating toward 10% FBS. Bottom-side fluorescence image segmentation mask (yellow) is blended with phase-contrast and fluorescence images acquired for NucLight Red HT-1080 cells migrating toward 10% FBS taken at t=0, 24, and 48 hours. Cells were seeded at 500 cells per well into an IncuCyte™ ClearView 96-Well Cell Migration Plate.

Chemotaxis adherent protocol Cyto D response

Cytochalasin inhibition of NucLight Red chemotaxis. NucLight Red HT-1080 cells were pre-treated with varying concentrations of Cytochalasin D prior to the addition of 10% FBS chemoattractant into the ClearView reservoir plate. Images were analyzed for the cells that migrated to the bottom-side of the membrane and counted (n=4 per condition).

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Detailed Non-Adherent Chemotaxis Cell Migration Protocol

Detailed Non-Adherent Chemotaxis Cell Migration Protocol

A detailed demonstration protocol designed to enable you to run a successful IncuCyte™ Chemotaxis Cell Migration Assay with non-adherent human neutrophils.

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Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte™ Chemotaxis Cell Migration Assay. Note, that the protocol does not include a description of any experiments included for optimizing seeding density or surface coatings. Here we specifically describe the use of the IncuCyte ZOOM® instrument for establishing and quantifying a chemotactic response of neutrophils toward fMLP.



Neutrophil Chemotaxis

Materials

Freshly isolated neutrophils

RPMI-1640 + 0.5% Human Serum Albumin (Note: BSA CANNOT be used in replacement of HSA. We have found that neutrophils will not migrate toward C5a or IL-8 if BSA is used as the serum albumin)

RPMI-1640 (ATCC 30-2001)

HSA (Sigma-Aldrich A9731-5G)

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

2x IncuCyte™ ClearView 96-Well Cell Migration Reservoir Plate (Essen 4600 or 4601)

Matrigel® Matrix (Corning 354234)

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

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

fMLP (Sigma-Aldrich F3506-10MG)

Protocol

NOTE: All steps, other than the initial polymerization of the coating matrix, are performed at ambient temperature and using standard sterile cell culture technique prior to placing the cell migration plate into the IncuCyte ZOOM® instrument for imaging.

1.Coat both sides of the membrane with 50 µg/mL Matrigel® + 10% FBS adding 20 µL to the insert wells (reverse pipette) and 150 µL to the reservoir wells (pre-fill reservoir and gently place the insert into the reservoir plate containing coating matrix). In this case, a second reservoir plate will be loaded with chemoattractant and used during the experiment. Learn how to coat
NOTE: The cell migration plate and all reagents, must be pre-chilled to 4°C. We recommend using a CoolSink™ to keep the plate cold during the coating procedure.

2.Place the ClearView Cell Migration plate at 37° C and incubate for 30 minutes.

3.Remove the ClearView plate from 37° C and allow to cool down to ambient temperature for 30 minutes.
NOTE: This step is important in order to achieve uniform cell distribution within each well.

4.Perform a cell count (e.g. trypan blue staining + hemacytometer). Centrifuge the cell suspension (1000 RPM, 4 minutes) and resuspend the cell pellet in culture medium at 83,333 cells per mL.
NOTE: When studying inhibitors of cell migration, cell seeding volume is reduced to 40 µL per well, and the cell seeding stock is increased to 125,000 cells per mL. This allows for 20 µL addition of test reagent.

5.Immediately prior to neutrophil addition, aspirate Matrigel® Matrix coating from both the reservoir plate and insert. Add 200 µL D-PBS to the reservoir plate then gently place the insert into the reservoir.

6.Using a manual multi-channel pipette and reverse pipetting technique, seed cells (60 µL per well, 5,000 cells per well) into every well of the insert plate. Calculation: 83,333 cells/mL x 0.06 mL = 5,000 cells per insert well. how to seed

7.In order to eliminate bubbles that may be present on the membrane surface, after dispensing cells, use a manual multi- channel pipette and set at 40 µL and triturate wells. Learn how to remove bubbles

8.Allow the neutrophils to settle at ambient temperature on a level surface for 45 – 60 minutes.

9.During cell settling, prepare fMLP chemoattractant dilutions.

10.Using a manual multi-channel pipette, add 200 µL of the chemoattractant and control medium to the appropriate wells of the second reservoir Plate.

11.Carefully transfer the insert plate containing cells into the pre-filled second reservoir plate containing medium ± chemoattractant. Learn how to transfer

12.Place the IncuCyte™ 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.

13.In the IncuCyte ZOOM® software; schedule 24 hour repeat scanning (10x) for every 30 minutes. NOTE: This schedule is only for scanning a single plate. Fewer scans will be included if scheduling multiple plates.

a. Objective: Ensure 10x objective is installed.

a. Vessel Type: Select "ClearView Cell Migration"

a. Channel Selection: Select "Phase"

a. Scan Mode: Select "Chemotaxis (Top/Bot)" scan type and desired Scan Pattern

Note: IncuCyte™ instrument estimates a scan time of 20 minutes per plate (phase only); however, the actual scan time can take longer if condensation is not wiped away.

Example of Data Generated with the Detailed Non-Adherent Chemotaxis Cell Migration Protocol
Chemotaxis non-adherent protocol representative images

Representative images of neutrophils migrating toward 50 nM fMLP. A top-side segmentation mask (yellow) is blended with original phase contrast images of freshly isolated neutrophils migrating toward 50 nM fMLP taken at t=0, 2 hours, and 4 hours. Cells were seeded at 5,000 cells per well into an IncuCyte™ ClearView 96-Well Cell Migration Plate.

Chemotaxis non-adherent protocol fMLP response

Kinetic analysis of neutrophils migrating toward fMLP. Freshly isolated neutrophils were seeded on a coated ClearView insert (50 μg/mL Matrigel® Matrix + 10% FBS) and allowed to settle at ambient temperature for 1 hour. The insert was then placed into a reservoir containing decreasing dilutions of the fMLP. Total neutrophil area normalized to max area is plotted (n=4 per condition).

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