Chemistry 532: Key to Homework 1

Mammalian Cell Culture and Flow Cytometry

The spleen and lymph nodes from two healthy mice were harvested for lymphocytes. The cells were bead-sorted for CD4+ T cells to give 4 x 10⁷ cells. The cells were split into two aliquots with 20 mL of media each. In the first flask Mitogen A and growth factors were added and, in the second flask, Mitogen B and growth factors were added (the identities of mitogens and growth factors are irrelevant for the following questions).

At 72 h 1 mL of cell suspension was removed from each flask and the suspension was plated at 100 µL/well and 1 µCi of ³H-thymidine was added to each well. At 96 h the wells were harvested and counted; the average counts for cells taken from the first flask (Mitogen A) was 2100 cpm and the average counts for cells taken from the second flask (Mitogen B) was 43,000 cpm. The number of cells in each flask was also counted at 96 h using a hemocytometer with Trypan Blue. The number of viable cells present in the first flask was 0.8 x 10⁶ cells/mL and the number of cells in the second flask was 0.7 x 10⁴ cells/mL.

Lymphocytes harvested from a healthy organism are resting (i.e., they are in G₀). Some mitogens can cause lymphocytes to advance into cell cycle and then proliferate. In some cases they can be transformed and, thus, will continue to proliferate. It is also not uncommon for primary lymphocytes to initially proliferate and then die off. In general, cells in G₀ that have not proliferated by 72-96 h will have either died or are in the process of dying (i.e., they will uptake Trypan Blue). For example, a large number of cells in the second flask (Mitogen B) have died as expected. However, proliferation measured as ³H-thymidine uptake is also observed with these cells. On the other hand, the cells in the first flask do not appear to have died but, proliferation is not detected by ³H-thymidine uptake.

Question: There are several possible scenarios that could produce the observations made for each flask at 96 h. Describe at least two scenarios for what is happening in each flask and give a detailed set of experiments that you could do to discriminate between the possible scenarios. Your experiments should describe what assays you would use, when you would use them, and how the results would be interpreted.

There are many possible scenarios for each situation. These are only my suggestions of what are the most obvious (to me at least).

 Flask A- Scenario 1: The mitogen could have stimulated a progresion of cell cycle that then arrested prior to mitosis. The number of viable cells would be similar to the start and the cells would have to been arrested either before or after S-phase prior to 72 h. At the 96 h time-point some of the cells could have been removed, stained with PI using detergent, and analyzed by flow cytometry for cell cycle. It would be anticipated that most of the cells would show a DNA content consistent with either G1- or G2-phase. The cells could have also been analyzed for cell cycle at earlier time-points to assay for cell-cycle progression.

 Flask A- Scenario 2: The mitogen could have stimulated some or most of the cells to proliferate and, at some point prior to 72 h they had arrested. By 96 h many of the cells had died and the observation of a similar number of cells as at the start is purely coincidental. At the beginning of the experiment some of the cells could have been labeled with CFSE and, at 96 h, the remaining cells could have been analyzed by flow cytometry. If the cells had proliferated there would be a much reduced concentration of CFSE in the remaining cells. In addition, the cells could have been analyzed for proliferation by tritiated-thymidine uptake at earlier time-points.

 Flask B- Scenario 1: The mitogen could have stimulated only a few of the cells to proliferate while most died. At 72 h the small population of cells that are proliferating will take up tritiated thymidine even though the number of cells counted at 96 h is still small. At the beginning of the experiment some of the cells could have been labeled with CFSE and, at 96 h, the remaining cells could have been analyzed by flow cytometry. If some of the cells are proliferating they would have a much reduced concentration of CFSE. One could have also counted the number of viable and non-viable cells at much earlier time-points by Trypan Blue exclusion or with PI staining and flow cytometry.

 Flask C- Scenario 2: The mitogen could have stimulated all of the cells to proliferate but they did so at very different rates and they only went through several mitotic cycles each. Thus, by 72 h most had proliferated and were either dead or dying. A few of the cells that were slower to start were still proliferating at 72 h and were still alive at 96 h. The major difference between this scenario and the one directly above is whether or not the majority of the cells actually proliferated. This could be assayed by tritiated thymidine uptake at earlier time points in addition to counting viable vs non-viable cells at the earlier time-points.

 Note: The last scenario is what would normally happen when using any conventional mitogen.