상단으로 이동
Products Applications Publications

IncuCyte™ Protocols : Immuno-Oncology


IncuCyte™ Immune Cell Killing General Protocol

IncuCyte™ Immune Cell Killing General Protocol

This protocol provides an overview of the IncuCyte™ Immune Cell Killing Assay methodology which can be used with adherent or suspension tumor cells and your choice of immune effector cells.

더 보기 View Information


General Demonstration Protocol

Immune cell recognition and killing of unwanted target cells, such as emergent tumor cells, is a critical component of the human host defense mechanism. Antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell killing are two mechanisms of cellmediated immune response. Each of these processes involves the stimulation of immune cell sub-populations, such as natural killer (NK) cells or T lymphocytes, which then actively lyse target cells. Understanding the interplay between immune and cancer cells and restoring and promoting the immune system’s capacity to fight and eliminate tumors (“cancer immunotherapy” or “immuno-oncology”) is an exciting and promising research field. Common methods used to assess immune cell killing of cancer cells are often end point, include cell lifting (e.g., flow cytometry), measure indirect readouts of tumor cell viability (e.g., LDH, GAPDH release assays) or immune cell activation (ELISpot), incorporate radioactivity (51Cr release), and are generally non–image-based. We have developed a new image-based co-culture methodology that combines direct measurements of tumor cell death with nowash, mix-and-read protocols suitable for screening.

The assay enables:

Real-time visualization and fully automated analysis of tumor cell killing

Direct measurements of tumor cell death and viability

Visualization of the interplay between immune cells and tumor cells via time-lapse movies

Insight into events leading up to cytotoxicity (e.g., cytotoxic lag times, morphological changes of effector cells)

fig_cell-killing-general-protocol_1

Figure 1. IncuCyte™ Immune Cell Killing assay concept. Target tumor cells in culture with your choice of immune cells (e.g., T cells or PBMCs) are repeatedly imaged and analyzed within the stable environment of your cell culture incubator using the IncuCyte™ live-cell imaging system. Tumor cell death is measured directly and in real time using the mix-and-read IncuCyte™ Caspase 3/7 reagent, a substrate that is cleaved during target cell apoptosis to release a green-fluorescent DNA dye that stains the nuclear DNA. IncuCyte™ image analysis software enables automated detection and selective quantitation of tumor cell death in real time.



Immune cell killing of adherent tumor cells

This protocol provides an overview of the IncuCyte™ Immune Cell Killing Assay methodology for adherent target tumor cells. It is compatible with the IncuCyte ZOOM® instrument using your choice of immune cells and treatments and supports the use of nonlabeled or nuclear-labeled target tumor cells. The flexible assay format is suitable for cytotoxic T cell killing and antibody-dependent cell-mediated cytotoxicity (ADCC) assays.

Protocol_immuno-oncology-general

General Protocol

Day 0:

1.Seed target cancer cells (100 μL per well) at an appropriate density into a 96-well flat-bottom plate (Corning 3595) such that by day 1 the cell confluency is approximately 20%. The seeding density will need to be optimized for each tumor cell line used; however, we have found that 1,000 to 3,000 cells per well are reasonable starting points.

Target cell growth can be monitored using the IncuCyte ZOOM® live-cell imaging device and confluence algorithm.

Optional: Target cells can be labeled with NucLight™ Red live-cell labeling reagent (Essen BioScience 4476) to enable simultaneous real-time counting of viable tumor cells.

Day 1:

1.Once the target cells have reached appropriate confluency, remove the cell plate from the incubator, remove the medium and replace it with medium containing the IncuCyte™ Caspase-3/7 Apoptosis Reagent (Essen BioScience 4440): 25 μL, prepared at 4x final assay concentration and test materials (e.g., T cell stimuli, antibodies, cytokines; 25 μL, prepared at 4x final assay concentration).

2.Seed 50 μL of your chosen effector cells (e.g., T cells, PBMCs) into the appropriate wells of the cell plate to achieve a total assay volume of 100 μL. It is recommended that different target-to-effector cell ratios are tested (e.g., 1:5, 1:10).

3.Remove any bubbles from the cell plate and place into the IncuCyte ZOOM® instrument. Allow the plate to warm to 37°C for 20 minutes.

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.

4.Image the plate in the IncuCyte ZOOM® instrument with a 10x objective using the Standard Scan Type. We recommend 2 images per and scanning every 3 hours.

닫기 Close Panel

Detailed Immune Cell Killing Protocol for Adherent Target Cells

Detailed Immune Cell Killing Protocol for Adherent Target Cells

A detailed demonstration protocol designed to enable you to run a successful IncuCyte™ Immune cell killing Assay with adherent SK-OV-3 ovarian cancer target cells and PBMC immune cells

더 보기 View Information


Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte™ Immune Cell Killing Assay using adherent target cells. Note that the protocol does not include a description of any experiments included for optimizing seeding density or target: effector cell ratios. Here we specifically describe the use of the IncuCyte ZOOM® instrument for establishing and quantifying the killing of SK-OV-3 ovarian cancer cells by anti CD3 antibody/IL-2 activation of the T cell subpopulation of peripheral blood mononuclear cells (PBMCs).

Materials

Target cells: SK-OV-3 ovarian cancer cells

Optional—tumor cells can be labeled with NucLight™ Red (Essen BioScience 4476) to provide a simultaneous count of viable target cells.

Growth medium: McCoy’s 5a medium + 15% FBS + 1% Glutamax

EEffector cells: Peripheral blood mononuclear cells (fresh or frozen, e.g., Cambridge BioScience CTL-PBMC/SER-PBMC)

96-well flat-bottom microplate (Corning 3595)

Assay medium: RPMI 1640 (Life Technologies 11875-085) + 10% FBS

IncuCyte™ Caspase 3/7 Reagent (Essen BioScience 4440)

Anti-CD3 antibody (eBioscience 16-0037) + rh IL-2 (PreproTech 200-02)

0.25% Trypsin/EDTA (Life Technologies 25200)

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



Protocol

Day 0:

1.Remove serum-containing medium from SKOV-3 target cell culture and gently rinse twice with D-PBS.

Note: Culture should be at 75–80% confluence in a T-75 flask.

2.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 10,000 cells/mL.

3.Using a manual multichannel pipette, seed cells (100 μL/well, i.e., 2000 cells/well) into every well of a flat-bottom microplate.

4.Let the plate stand at ambient temperature for 30 min. Position the plate into the IncuCyte ZOOM® live-cell imager and leave for 20 min to equilibrate before scheduling the first scan.

5.Schedule 24-hr repeat scanning (10x objective) for every 3 hr, with the first scan to commence immediately. Monitor cell confluence for the next 18 hr (overnight) until the desired confluence (e.g., 20%) is achieved.

Day 1:

1.On the morning of the assay prepare a 10 μM solution of the IncuCyte™ Caspase 3/7 apoptosis green fluorescence detection reagent (Essen BioScience 4440) (4x final assay concentration of 2.5 μM) in assay medium. Warm to 37°C in an incubator.

2.Prepare the anti-CD3 antibody (100 ng/mL) + IL-2 (10 ng/mL) T cell activator treatment at 4x final assay concentration in assay medium and warm to 37°C.

3.Remove the target cell plate from the incubator. Aspirate the medium, taking care not to damage the cell layer. Using a multichannel pipette, transfer 25 μL of the warmed caspase 3/7 solution into each well. Then transfer 25 μL of the warmed anti-CD3 antibody + IL-2, or vehicle, into the appropriate wells of the cell plate.

4.Add an additional 50 μL medium containing the effector cells (PBMCs, see below) to form a total assay volume of 100 μL.

Note: It is advised that some control wells containing only target cells are included (+ vehicle and + PBMC activators).

5.Remove any bubbles from all wells by gently squeezing a wash bottle (containing 100% ethanol with inner straw removed) to blow vapor over the surface of each well. Keep the tip of the wash bottle approximately 5 cm from the surface of the medium.

6.Position the de-bubbled cell plate in the IncuCyte ZOOM® instrument and allow it to equilibrate for 20 min prior to the first scan. Schedule 24-hr repeat scanning (10x) for every 2–3 hr for up to 5 days.

Objective: 10x,

Vessel Type: Corning 3595

Scan Mode: Standard

Scan Pattern: 2 images per well

Channel: Phase + “Green” (+“Red” if NucLight™ Red target cells are used)


Preparation of PBMCs

The target cell:PBMC ratio for this protocol is 1:5. It is recommended that more than one ratio is tested in order to observe optimum levels of target cell death.

1.If using frozen PBMCs, place cells in a water bath at 37°C and once thawed, remove the contents and gently transfer to a 50 mL Falcon tube. If using fresh PBMCs, remove the contents and gently transfer to a 50 mL Falcon tube.

2.Slowly add warmed assay medium to the cell suspension to make a total volume of 40 mL, gently agitating at the same time.

3.Centrifuge the cell suspension (1000 RPM, 10 min) and resuspend first using a 1 mL pipette to gently break up the pellet, and then a stripette to add additional medium included to achieve an appropriate density for counting (e.g., 1 x 106 cells/mL).

4.Perform a cell count (e.g., trypan blue staining + hemacytometer) and resuspend the cells at 1 x 105 cells/mL.

5.Using a manual multichannel pipette, transfer cells into chosen wells of the cell plate (50 μL/well, i.e., 5 x 103 cells/well).


Analysis

Target-cell apoptosis is quantified in the IncuCyte™ software by counting the total number of “large” green-fluorescent objects (nuclei) in the field of view over time. Proliferation of target cells is measured from the red object count, corresponding to the number of red cell nuclei. Data are expressed as the number of fluorescent objects per mm2. To generate these metrics, the user must create a Processing Definition and Analysis Job, as described within the IncuCyte ZOOM® Fluorescent Processing Overview Technical Note, which are suited to the cell type, assay conditions, and magnification. Note that caspase 3/7–positive effector cells will also fluoresce. Analysis filters (area, mean intensity, eccentricity) should be applied to exclude these cells (and thus isolate the target cell signal) from the analysis.

Example data generated with the Detailed Immune Cell Killing Protocol for Adherent Target Cells
fig-1_cell-killing_adherent-protocol

Figure 1. T-cell killing. Representative blended phase contrast and green-fluorescent images (10x) of SK-OV-3 and PBMC co-cultures in the presence of IncuCyte™ Caspase 3/7 Reagent at 96 hr in non-activated PBMCs (control; A, B) and activated PBMCs (anti-CD3 antibody [100 ng/mL] + IL-2 [10 ng/mL]) (C, D). In each case, the right-hand panel shows the masking (yellow) of apoptotic target (but not effector) cells with the IncuCyte™ processing definition. Note the induction of target-cell apoptosis in the anti-CD3 antibody/IL-2–treated co-cultures. Panel (E) shows an IncuCyte™ software view of the time-course of target-cell caspase 3/7 apoptotic cell (green object) count with non-activated PBMCs (blue) and activated PBMCs (anti-CD3 antibody [100 ng/mL] + IL-2 [10 ng/mL]) (pink) co-cultures. Note the onset of apoptosis in the treatment group at approximately 20 hr.

fig-2_cell-killing_adherent-protocol

Figure 2. T-cell killing and inhibition of proliferation.

Representative blended phase contrast and red-fluorescent images (10x) of NucLight™ Red labeled SK-OV-3 and PBMC co-cultures in the presence of IncuCyte™ Caspase 3/7 substrate at 96 hr in non-activated PBMCs (control; A, B) and activated PBMCs (anti-CD3 antibody [100 ng/mL] + IL-2 [10 ng/mL]) (C, D). In each case, the right-hand panel shows the masking of target-cell nuclei (blue) with the IncuCyte™ processing definition. Note the inhibition of target-cell proliferation in the anti-CD3 antibody/IL-2–treated co-cultures. Panel (E) shows an IncuCyte™ software view of the time-course of target-cell proliferation (red object count) with non-activated PBMCs (blue) and activated PBMCs (anti-CD3 antibody [100 ng/mL] + IL-2 [10 ng/mL]) (pink) co-cultures. Note the onset inhibition of proliferation at approximately 20 hr.

닫기 Close Panel

Detailed Immune Cell Killing Protocol for Suspension Target Cells

Detailed Immune Cell Killing Protocol for Suspension Target Cells

A detailed demonstration protocol designed to enable you to run a successful IncuCyte™ Immune cell killing Assay with suspension WIL2-NS B lymphocytes and PBMC immune cells

더 보기 View Information


Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte™ Immune Cell Killing Assay using suspension target cells. Note that the protocol does not include a description of any experiments included for optimizing seeding density or target:effector cell ratios. Here we specifically describe the use of the IncuCyte ZOOM® instrument for establishing and quantifying the killing of fluorescently labeled WIL2-NS B lymphocyte cells by activated peripheral blood mononuclear cells (PBMCs).

Materials

Target cells: WIL2-NS B-lymphocyte cells pre-transduced with NucLight™ Red (nuclear red fluorescent red fluorescent label, Essen BioScience 4476)

Growth medium: RMPI 1640 (Life Technologies 11875-093) + 10% FBS + 0.5 μg/mL Puromycin

Assay medium: RMPI 1640 (Life Technologies 11875-093) + 10% FBS

Effector cells: Peripheral blood mononuclear cells (fresh or frozen, e.g., Cambridge BioScience CTL-PBMC/SER-PBMC)

96-well round-bottom ultra-low attachment microplate (Corning 7007)

Anti-CD3 antibody (eBioscience 16-0037) + rh IL-2 (PreproTech 200-02)

0.25% Trypsin/EDTA (Life Technologies 25200)

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



Protocol

1.Prepare anti-CD3 antibody (100 ng/mL) + IL-2 (10 ng/mL) T cell activator treatment at 4x final assay concentration in assay medium at ambient temperature. Transfer 50 μL of the activator treatment (or medium) into selected wells of the 96-well assay plate using a manual multichannel pipette.

2.Remove a T-75 flask of WIL2-NS NucLight™ Red (target) cells (2 x 105–1 x 106 cells/mL) from the tissue culture incubator and place within a sterile cell culture hood. Transfer the cell suspension into a 50 mL Falcon tube and centrifuge (1000 RPM, 4 min).

3.Aspirate the medium and gently resuspend the pellet with 500 μL Trypsin/EDTA using a 1 mL pipette (addition of trypsin avoids cell clumping). Incubate for 2 min at 37°C. Add 9 mL of assay medium and resuspend the cells.

4.Perform a cell count (e.g., trypan blue staining + hemacytometer). Centrifuge (1000 RPM, 4 min) the cells and resuspend the pellet in assay medium (5 x 103 cells/mL).

5.Transfer the target cells into all wells of the round-bottom ultra-low attachment plate (50 μL/well, i.e., 250 cells/well). Leave the cell plate in the hood at ambient temperature for 30 min to allow the cells to settle.

6.Prepare the effector cells (PBMCs)—see below. Transfer the cells into chosen wells of the cell plate (100 μL/well, i.e., 2.5 x 103 cells/well).

Note: It is advised that some control wells containing only target cells are included (+ vehicle and + PBMC activators).

7.Remove bubbles from all wells by gently squeezing a wash bottle (containing 100% ethanol with inner straw removed) to blow vapor over the surface of each well. Keep the tip of the wash bottle approximately 5 cm from the surface of the medium.

8.Place the de-bubbled cell plate into an IncuCyte ZOOM® instrument and allow it to equilibrate for 20 min prior to scheduling the first scan.

9.Schedule 24-hr repeat scanning (10x) for every 2–3 hr for up to 5 days, with the first scan to commence immediately.

Objective: 10x

Vessel Type: Corning 7007

Scan Mode: Standard

Scan Pattern: 1 image per well

Channel: Phase, Red


Preparation of PBMCs

The target cell:PBMC ratio for this protocol is 1:10. It is recommended that more than one ratio is tested in order to observe optimum levels of target cell death.

1.If using frozen PBMCs, place cells in a water bath at 37°C and once thawed, remove the contents with a 2 mL stripette and gently transfer to a 50 mL Falcon tube. If using fresh PBMCs, remove the contents with a 2 mL stripette and gently transfer to a 50 mL Falcon tube.

2.Slowly add warmed assay medium to the cell suspension to make a total volume of 40 mL, gently agitating at the same time.

3.Centrifuge the cell suspension (1000 RPM, 10 min) and resuspend first using a 1 mL pipette to gently break up the pellet, and then a stripette to add additional medium included to achieve an appropriate density for counting (e.g., 1 x 106 cells/mL).

4.Perform a cell count (e.g., trypan blue staining + hemacytometer) and resuspend the cells at 2.5 x 105 cells/mL.

Analysis

The number of target cells is quantified using the following metrics:


1.Red mean image fluorescence (MIF, found under the IncuCyte™ default Scan Metric Selection)—a metric that describes the fluorescence intensity for each pixel within the entire image and calculates the mean value. Although not a direct measurement of target cell population size, MIF is a proven useful surrogate. The metric includes no image analysis and eliminates the need to mask low-intensity cells, which can be challenging.

2.Red fluorescence area (μm2/image)—follow the instructions provided within the IncuCyte ZOOM® Fluorescent Processing Overview Technical Note to (1) create a Processing Definition to mask target cells using fluorescent object analysis; and (2) launch an Analysis Job to analyze images and produce metrics based on parameters contained within the saved Processing Definition. Use the “Fixed Threshold” parameter to accurately mask the spheroid (e.g., a recommended fixed threshold of 8 RCU for WIL2-NS NucLight™ Red cells).

Example data generated with the Detailed Immune Cell Killing Protocol for Suspension Target Cells
fig-1_protocol_cell-killing_suspension

Figure 1. Representative phase contrast and fluorescence overlay images (10x) at 120 hr of (A) WIL2-NS with non-activated PBMC co-culture and (B) WIL2-NS with activated PBMCs co-culture (anti-CD3 antibody [100 ng/mL]; IL-2 [10 ng/mL]). Time-course (C) of proliferation of WIL-2NS NucLight™ cells in co-culture with either non-activated PBMCs (blue) or activated PBMCs (anti-CD3 antibody/IL-2, 10 ng/mL, pink).

닫기 Close Panel