Flow cytometric quantification of all phases of the cell cycle and apoptosis in a two-color fluorescence plot

Abstract

An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.

Figure 1. Cell cycle analysis of human KG1a leukemic cell line exposed to cell cycle modifiers.

The cells were stained with 7-amino-actinomycin D (7-AAD) and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor®488. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. (Left) Effects of camptothecin (1 µM, 6 h, 37°C, 5% CO₂). The histograms present the proportions of apoptosis and all cell cycle phases normalized to those of the untreated leukemic cells. (Right) Effects of contact with bone marrow primary mesenchymal stromal/stem cells (MSCs) (72 h, 37°C, 5% CO₂). The leukemic cells were identified by concomitant staining with APC-Cy7-conjugated anti-CD45 mAb. The histograms present the proportions of apoptosis and all cell cycle phases normalized to those of the leukemic cells without MSCs. (representative experiment).

Figure 2. Cell cycle analysis of human MV4–11 leukemic cell line exposed to AZD8055.

The cells were stained with 7-AAD and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor®488. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. Effects of AZD8055 (10 nM and 100 nM, 24 h, 37°C, 5% CO₂). The results present the percentage of cells in the apoptosis and all cell cycle phases. (representative experiment).

Figure 3. Cell cycle analysis of human lymphocytes exposed to PHA.

The cells were stained with 7-AAD and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor®488. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. Effects of PHA (170 µg/mL, 72 h, 37°C, 5% CO₂). The results present the percentage of cells in the apoptosis and all cell cycle phases. (representative experiment).

Figure 4. Cell cycle analysis of human KG1a leukemic cell line exposed to colcemid.

The cells were stained with 7-AAD and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor®488. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. Effects of colcemid (0.1 µg/mL, 30 min and 1 h, 37°C, 5% CO₂). The results present the percentage of cells in the apoptosis and all cell cycle phases. (representative experiment).

Figure 5. Cell cycle analysis cell subpopulations in mixed B and T cell suspension (70% Raji and 30% Jurkat cells).

The cells were stained with 7-AAD, Alexa Fluor®488-conjugated anti-Ki67, Alexa Fluor®488-conjugated anti-phospho(Ser10)-histone H3 and Horizon™ V450-conjugated anti-CD3 antibodies. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. Lymphocytes were identified by CD3/FSC gating. (representative experiment).

Figure 6. Cell cycle analysis of bone marrow cells of patient suffering from AML.

The cells were stained with 7-AAD, Alexa Fluor®488-conjugated anti-Ki67, Alexa Fluor®488-conjugated anti-phospho(Ser10)-histone H3 and APC-Cy7-conjugated anti-CD45 antibodies. An isotype control staining with Alexa Fluor®488 mouse IgG1 was performed. The leukoblasts, lymphocytes and granulocytic-lineage cells were identified by CD45/SSC gating. (representative analysis).