Materials and Methods
Acridine orange (AO) and propidium iodide (PI) are nucleic acid binding dyes that can be used to measure the cell viability. Both AO and PI are considered skin irritants and may be harmful if ingested or inhaled; they are currently not classified as carcinogenic. Since AO is cell permeable, all stained nucleated cells generate a green fluorescence. PI (~668 Daltons) only enters cells with compromised membranes and therefore dying, dead, and necrotic nucleated cells stained with PI generate a red fluorescence. When cells are stained with both AO and PI, live nucleated cells only fluoresce green and dead nucleated cells only fluoresce red. This is due to Förster resonance energy transfer (FRET); where the PI signal absorbs the AO signal producing no spill-over or double positive results. Additionally, other membrane-exclusion viability dyes such as: ethidium bromide (EB), 7AAD, SYTOX green/red, DRAQ5 and others may also be used instead of PI. Cell viability is calculated by examining the ratio of the number of live to the number of dead fluorescing cells. This assay can not only be used to measure the viability of nucleated cells in cell culture and purified samples but also in complicated samples such as PBMC, whole blood, bone marrow, bronchoalveolar lavage, tumor digests, primary samples and many more.
Jurkat cell preparation
Jurkat cells were cultured in RPMI-1640 Medium. For a viability comparison of room temperature-incubated cells, one flask was taken out of the incubator and placed into a bench-top drawer. The flask was kept at room temperature for the remainder of the experiment. A small aliquot of cells was removed from the flask at the following time points: 0, 6, 12, 24, 48, 72, 96, and 168 h. A similar experiment was conducted to study the effect of different TB concentrations on Jurkat cell viability, which was measured at the following time points: 0, 3, 6, 9, 12, 27, and 33 h. (The time points were shorter than the first experiment because it was targeted towards the time frame where the viability difference, shown here in Figure 2, was most apparent.) For a heat shock viability comparison, Jurkat cells were placed in boiling water for 15 min. After heat shock, five samples were prepared at viabilities of 0, 25, 50, 75, and 100% by mixing the heat-killed and fresh Jurkat cells at the appropriate volumes.
Time-course viability of room temperature-incubated Jurkat cells
The viability and concentration of the room temperature-incubated Jurkat cells were determined and compared using four detection methods: (1, 2) measuring viability staining with PI or AO/PI and counting with Cellometer Vision, (3) staining with 0.4% TB and counting with Cellometer AutoT4, and (4) staining with 0.4% TB and manual counting with hemacytometer. The AO/PI staining procedure was performed as previously described . Room temperature-incubated cell samples at 0, 6, 12, 24, 48, 72, 96, and 168 h were mixed with PI, AO/PI, or TB, and analyzed immediately with Cellometer Vision, AutoT4, and hemacytometer. The viability and concentration were measured in quadruplicate for all time points.
Trypan blue concentration-dependent viability comparison
The effect of TB concentration on viability of room temperature-incubated Jurkat cells was determined and compared using two detection methods: (1) measuring viability by AO/PI and counting with Cellometer Vision, and (2) staining with various concentrations of TB and manual counting with hemacytometer. Each room temperature-incubated cell sample at 0, 3, 6, 9, 12, 27, and 33 h was mixed with AO/PI or 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125% of TB and analyzed immediately with Cellometer Vision and hemacytometer. The viability and concentration were measured in quadruplicate for all time points.
Heat shock viability comparison
Freshly collected Jurkat cells were separated into two equal aliquots, one was heat shocked by boiling the samples for 15 min (100% non-viable) and the other was not (100% viable). The cells from these two aliquots were then mixed at different ratios to produce cell suspensions. The viability and concentration of each heat-shocked/live viability mixture (0, 25, 50, 75 and 100%) was determined and compared using three detection methods: (1) Measuring viability staining with PI and counting with Cellometer Vision, (2) staining with 0.4% TB and counting with Cellometer AutoT4, and (3) staining with 0.4% TB and manual counting with hemacytometer. Each heat-shock/live mixture sample was mixed with PI or TB, and analyzed immediately with image cytometer and hemacytometer. The viability and concentration for each of the three independent experiments were measured in quadruplicate for all five mixtures.
Image cytometry automated cell-counting protocol
Jurkat cells stained with PI, AO/PI, or TB were pipetted immediately after mixing into a Nexcelom disposable counting chamber. The Cellometer Vision utilizes fluorescence (FL) optics modules of VB-535- 402 (pseudo-color green) and VB-660-502 (pseudo- color red) to detect AO and PI, respectively. The Cellometer AutoT4 uses a color camera to detect TB stained Jurkat cells. For TB image analysis, live cells with bright center and dark cells stained with TB in the brightfield (BF) were enumerated. For PI image analysis, total cells in the BF and dead cells in the FL channel were enumerated. For AO/PI image analysis, AO-stained live cells and PI-stained dead cells in green and red FL channels were enumerated.
Hemacytometer manual cell counting protocol
Jurkat cells stained with TB were pipetted immediately into a Neubauer hemacytometer. Live and dead cells were enumerated manually under light microscopy for samples from both controlled heat-shocked and room temperature-incubated Jurkat cells. Cell samples with an approximate 50% viability were used for manual counting.