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IncuCyte™ Protocols : Immune Cell Clustering & Proliferation


IncuCyte™ Immune Cell Clustering and Proliferation Protocol

Detailed Immune Cell Proliferation Protocol

This protocol is about Immune Cell Clustering and Proliferation

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Detailed Demonstration Protocol

The following protocol is a detailed example designed to enable you to run a successful IncuCyte™ Immune Cell Clustering and Proliferation Assay using peripheral blood mononuclear cells (PBMCs). Here we specifically describe the use of the IncuCyte ZOOM® instrument for establishing and quantifying the proliferation and clustering of the T cell subpopulation of PBMCs following activation with anti-CD3 antibody and IL-2.

Materials

Immune 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

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



Protocol

Day 1:

1.Prepare the anti-CD3 antibody (5–10 μg/mL) in PBS and the coat cell plate with 50 μL antibody.

2.Cover the plate with Parafilm and incubate at 37°C for 2 hr.

3.If using frozen PBMCs, place the 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.

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

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

6.Perform a cell count (e.g., trypan blue staining + hemacytometer) and resuspend the cell pellet in culture medium at 1 x 105 cells/mL.
Note: When studying inhibitors of immune cell proliferation, the cell seeding concentration is increased to 1.5 x 105 cells/mL and 100 μL is added to the cell plate following inhibitor addition. Inhibitors should be prepared at a 4 x final assay concentration and 50 μL added to wells prior to cell addition.

7.Prepare IL-2 (10 ng/mL) activation treatment at 4x final assay concentration and warm to 37°C.

8.Immediately prior to IL-2 addition, aspirate the anti-CD3 coating from the cell plate and wash twice with 200 μL PBS. Then transfer 50 μL IL-2 into the cell plate wells.

9.Using a manual multichannel pipette, seed the cells (150 μL/well, 15,000 cells/well) into every well of the cell plate.

10.Allow the immune cells to settle at ambient temperature on a level surface for 45 min.

11.Carefully place the cell plate into the IncuCyte ZOOM® instrument and allow the plate to warm to 37°C for 10 min.

12.In the IncuCyte ZOOM® software, schedule 24-hr repeat scanning (10x or 4x objective) for every 3–4 hr.

Objective: Ensure 10x or 4x objective is installed

Vessel Type: Corning 3595

Channel Selection: Phase

Scan Type: Standard

Scan Pattern: Select four images per well if using a 10x objective or one image per well if using a 4x objective


Analysis

Immune cell proliferation is quantified in the IncuCyte™ software using the confluence algorithm, as described within the IncuCyte ZOOM® Confluence Processing Technical Note. To quantify immune cell clustering, size filters are applied to the confluence algorithm to identify aggregates with a minimal area of 1500 μm2 and a maximal eccentricity (non-circularity) of 0.95. Results are expressed as the number of clusters per square millimeter.

Example data generated with the Detailed Immune Cell Clustering and Proliferation Assay Protocol
fig-1_proliferation-protocol

Figure 1. Representative images of PBMC proliferation. Phase-contrast images are overlaid with an IncuCyte ZOOM® confluence segmentation mask (yellow) of PBMCs activated with anti-CD3 antibody and IL-2. Images were taken at t=0 (A), 72 hours (B), and 144 hours (C). Cells were seeded at 15,000 cells/well. (D) Time course of PBMC proliferation in the presence of anti-CD3 antibody and IL-2, anti-CD3 antibody alone, and in the absence of activators.

fig-2_proliferation-protocol

Figure 2. Activation of PBMCs with anti-CD3/IL-2 induces T-cell aggregation. Representative phase-contrast images of PBMCs activated with anti-CD3 antibody and IL-2 were taken at t=0 (A), 72 hours (B), and 144 hours (C) and overlaid with the IncuCyte ZOOM® confluence segmentation mask (blue), indicating cell clusters with an area greater than 1500 μm2 and a maximal eccentricity (non-circularity) of 0.95. (D) Representative time courses of T-cell subpopulation clustering when activated with IL-2 (10 ng/mL) and decreasing concentrations of anti-CD3 antibody.

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