U937 cells often have large nuclei in the growth phase (being a monocyte rather than macrophage cell cell, they also tend to be about the same size as large T cell blasts - like Jurkats). Moving them to a lower serum, low glucose medium o/n before staining will help increase cytoplasm levels for vi ...

If you want to quantitate protein translocation, you'll need to use cell fractionation to obtain cytoplasmic and nuclear fractions. Newer imaging flow cytometers such as the Amnis ISX is another option. Cytospins or smears will just give you a qualitative picture.Use positively charged or lysine c ...

In many instances, the same Ab will work equally well. In other case, it might work better in solution with a cell suspension (e.g. for clow cytometry), or on a solid matrix (cells on a coverslip). Sadly, there is no way to empirically state that any given Ab will work - you just have to test it ou ...

To make killing curves for your drug, the ideal expt would be a series of conc of the drug (or solvent ctrl) added to wells previously seeded with a defined number of growth arrested cells (e.g. by serum starvation)(note that the drug conc expt itself can be done -/+serum, or in serum-free media to ...

Try the Dynal system then - thats column free, so that might translate to less stress. Post cyto enrichmnt, its easily 80+%.

The highest EDTA conc I have used has been 100 mM - but that has been to detach and maintain a single cell culture for a short period of time, without affecting surface receptors. You can perhaps try the PolyHEMA protocol outlined below - as you rightly said, eliminating Ca usually results in cell ...

Hi Grabin, Do you need very large numbers of cells (i.e why aren't you enriching for circ/exd macrophages rather than monocytes?)? Major contaminant cell populations (RBCs, platelets, lymphocytes, neuts) can be largely eliminated by Percoll or seq Ficoll gradients. DCs will be your smallest popu ...

Hi Asif - you need to specify amount of EDTA as a molar concentration (usually EDTA is made as a stock in water at 0.5Mat pH8, for cell culture, its diluted in PBS or HBSS). Also, cell type,, how long you want to grow them, and purpose for the adherent-->suspension switch. As you might have appre ...

We use the 9-layer porous flasks from Costar/Corning. Again, its fiddly, but the 550 ml of sups we get out of it has as much protein as 10 T225 flasks.

Hi Uzair, you can actually see apoptotic blebs under the microscope when the cells are stained with a Hoechst or DAPI or AO DNA dye. However, the process is NOT very quantitative, and requires a trained eye, ideally 'blinded' to experimental conditions for scoring. If you want to try a kinetic apop ...