Coulter EPICS XL-4 flow cytometer, Beckman Coulter GmbH, Germany, was used for analysis of cells by flow cytometry. Individual cells expressing different molecules can be detected by flow cytometry. Expression of pro- teins can be detected by staining cells with specific antibodies covalently conjugated with fluorochrome. Alternatively, cells expressing fluorescent proteins, such as GFP or YFP can be detected directly. When cells are placed on flow cytometer, a stream of cells is directed through an argon laser beam that excites fluorochrome to emit light. The emitted light is then detected by a photomultiplier tube specific for an emission wavelength of fluorochrome by virtue of a set of optical filters. The signal is amplified in its own channel and is displayed in a variety of different forms: histogram, dot blot or contour display.
Analysis of GFP and YFP expression by FACS
5 X 105 cells were pelleted down by centrifugation at 1000g for 2 minutes,
resuspended in 500 µl of fresh culture medium and used directly for flow cytometry.
Analysis of Flt3 surface expression by FACS
5X105 cells were pelleted down by centrifugation at 1000g for 2 minutes
and washed once with PBS, containing 0.2% FBS. 10µl of either anti-Flt3 antibody (clone SF 1.340, Immunotech, France) or isotype control antibody (mouse Ig G1antibody, Immunotech, France), both covalently conjugated to
phycoerythrin (PE), were added to the cells. Cells were mixed by flicking the tubes and incubated with the antibodies for 15 minutes at room temperature in the dark. 4 ml of PBS containing 0.2% FBS was then added to the cells. Cells were pelleted down by centrifugation at 1000g for 5 minutes, resuspended in 250µl of PBS containing 0.2% FBS, and analyzed by flow cytometry.
2.10.2 Determination of cell viability and numbers
When cells undergo necrosis or are at the late stages of apoptosis (secondary necrosis), they lose the integrity of the plasma membrane. These cells can be detected using exclusion dyes such as Trypan Blue. Trypan Blue stains only those cells, which have lost the membrane integrity, leaving cells with intact plasma membrane unstained.
counted using cell counting chambers. Cells which were negative for Trypan Blue staining were considered viable.
Cell growth assay with 32D cl.3 derived cell lines, expressing Flt3 ITD Cell growth reflects the rates of cell proliferation and apoptosis. Cell growth was determined by counting viable cells over a period of time.
Cell lines which were in culture for no more than 3 weeks were used for the assay. Prior to the start of the assay, all cell lines were sub-cultured as described in 2.7 in the presence of IL-3. Concentration of viable was determined using Trypan Blue dye staining as described above. Cells were washed 3 times with culture medium without IL-3, and resuspended in the culture medium with or without IL-3 to the final concentration of 1 X 104 viable cells/ml. Cells were plated in triplicates in 24 well plates at 1 ml of cell suspension per well. 72 hours after plating, concentration of viable cells was determined using Trypan Blue dye staining as described above.
2.10.3 Apoptosis assay using Annexin-V apoptosis detection kit
Annexin-V is a calcilum-dependent phospholipid binding protein, that has a great affinity for phosphatidylserine. In normal cells phosphatydilserine is present in the inner leaflet of the plasma membrane, while outer leaflet contains mostly neutral phospholipids. However, as cells undergo apopto- sis, loss of asymmetry in the plasma membrane phospholipids occurs. The amount of phosphatidylserine in the outer leaflet of the membrane increases. Therefore, Annexin-V binds to the surface of apoptotic, but not normal cells. Late stages of apoptosis (secondary necrosis) and necrosis, are accompanied by the loss of the integrity of the plasma membrane. Annexin-V can enter such cells and bind phosphatidylserine in the inner inner leaflet of cells. To discriminate between apoptotic and necrotic cells, staining with the vital dye 7-Amino-actinomycin (7-AAD) was performed in parallel to Annexin-V labeling.
Cells were pelleted by centrifugation, washed with PBS and resuspended in 100 µl of Annexin-V binding buffer at the concentration of 1 X 106 cells/ml. 2.5 µl of Annexin-V conjugated to PE and 2.5 µl of 7-AAD were added to the cells. Cells were mixed gently and incubated for 15 minutes at room temperature in the dark. 400 µl of binding buffer was then added to the cells and cells were analyzed by flow cytometry within 2-3 hours. All cells which were Annexin-V positive were considered apoptotic.
Apoptosis assay with 32D cl.3 derived cell lines expressing Flt3 wt Cell lines which were in culture for no more than 3 weeks were used for the assay. Prior to the start of the assay, all cell lines were sub-cultured as described in 2.7 in the presence on IL-3. Concentration of viable cells was determined using Trypan Blue dye staining as described above. Cells were washed 3 times with culture medium without IL-3, and resuspended in culture medium with or without 10% of WEHI-3B supernatant as a source of IL-3 or 100 ng/ml Flt3 ligand (Promocell, Germany) to the final concen- tration of 1X105 cells/ml. Cells were plated in triplicates in 24 well plates at 1 ml of cell suspension per well. Apoptosis was measured 24-48 hours after plating using Annexin-V apoptosis detection kit as described above.