Abstract

In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised non-viable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to label cells for mass cytometry determinations of live/dead ratios. In a one-minute incubation step, cisplatin preferentially labeled non-viable cells, from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for cytometry. Furthermore, the live/dead ratios were comparable between mass and fluorescence based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a one-minute “pulse” of cisplatin did not induce observable DNA damage or apoptotic responses even within 6 hours post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols.

Experiment Overview

Purpose: We sought to develop cisplatin as a versatile, user-friendly viability indicator for mass cytometry that would withstand a rigorous staining protocol including crosslinking, permeabilization, and multiple wash steps.
Samples were stimulated or inhibited with one or more of the following conditions: 125 uM PVO4, 20 uM etoposide
Surface and intracellular markers measured CD3, CD33, CD19, CD45, SLP-76 (BLNK) (pY128), H2AX (pS139), cleaved PARP, Cisplatin (as 195Pt)
Cytometer Used: DVS Sciences, Inc. CyTOF™ Mass Cytometer

Figure 1B: Cisplatin vs. LIVE/DEAD®

Aqua - Panel 1
Cells
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
Aqua - Panel 1
Cells
  • 579.2

Figure 1B: In a one-minute “pulse” staining protocol, cisplatin preferentially reacts with protein in dead cells. As a basis for comparison, an aliquot of partially heat-killed KG-1 cells was labeled with an amine-reactive fluorescent viability stain (Aqua).

Figure 1C: Cisplatin vs. LIVE/DEAD®

10uM Cisplat
50uM Cisplat
250uM Cisplat
Singlets
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
10uM Cisplat 50uM Cisplat 250uM Cisplat
Singlets
  • 1.6
  • 12.05
  • 1065.09

Figure 1C: Additional aliquots of partially heat-killed KG-1 cells were labeled with cisplatin at the concentrations shown and analyzed by mass cytometry.

Figure 2: Suspension and adherent cell lines

10 uM Cisplat
50 uM Cisplat
250 uM Cisplat
Jurkat
 
KG1
 
Ovcar
 
HeLa
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
10 uM Cisplat 50 uM Cisplat 250 uM Cisplat
Jurkat
  • 0.37
  • 7.55
  • 415.32
KG1
  • 0.04
  • 4.05
  • 290.25
Ovcar
  • 0.79
  • 20.92
  • 1754.2
HeLa
  • 2.29
  • 20.58
  • 1503.96

Figure 2: Cisplatin viability labeling was performed at 10, 50 and 250 μM final concentrations for suspension (Jurkat, KG-1) and adherent (HeLa, OVCAR3) cell lines. For all cell lines the percentage of dead cells was comparable as determined by mass cytometry and Trypan blue exclusion.

Figure 3: Reproducibility and dynamic range

Pt3(Pt195)Dd - Panel 2
Singlets
 
Replicate1
Replicate2
Replicate3
0% dead spiked in
 
1.25% dead spiked in
 
2.5% dead spiked in
 
5% dead spiked in
 
10% dead spiked in
 
20% dead spiked in
 
40% dead spiked in
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
Replicate1 Replicate2 Replicate3
0% dead spiked in
  • 2.21
  • 1.97
  • 1.56
1.25% dead spiked in
  • 2.5
  • 2.54
  • 3.14
2.5% dead spiked in
  • 3.72
  • 4.06
  • 3.98
5% dead spiked in
  • 6.52
  • 6.25
  • 5.69
10% dead spiked in
  • 10.77
  • 10.24
  • 11.17
20% dead spiked in
  • 19.27
  • 19.93
  • 20.57
40% dead spiked in
  • 34.6
  • 36.33
  • 36.13

Figure 3: Triplicate samples of HL-60 cells spiked with varying percentages of dead cells ranging from 0-40% were analyzed by mass cytometry using the cisplatin viability reagent. The average percent of dead cells in each sample with their standard deviation are shown. Mass cytometry measurements of cisplatin labeling reproducibly determined the percentage of dead cells over the whole analysis range.

Figure 4A: Cisplatin+ cells do not signal

All CD45+ cells
Myeloid
T cells
B cells
Mock stim - 10 mins
 
125 uM PVO4 - 10 mins
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
All CD45+ cells Myeloid T cells B cells
Mock stim - 10 mins
  • 4.61
  • 6.14
  • 2.18
  • 12.99
125 uM PVO4 - 10 mins
  • 3.19
  • 3.18
  • 5.07
  • 5.39

Figure 4A: Cryopreserved peripheral blood mononuclear cells were labeled with cisplatin, 25 μM, final concentration for one minute and treated with PVO4, 125 μM, final concentration for 15 minutes. An antibody that cross-reacted with phosphorylated SLP-76 (Tyr128) and SLP-65 (Tyr72) was used as an indicator of intracellular signaling.

Figure 4B Panel 1: Cisplatin+ cells do not signal

Singlets
Mock stim - 10 mins
 

Figure 4B Panel 1: To illustrate the potential confounding effect of non-viable cells in an assay of intracellular signaling, three hypothetical gating scenarios were compared. This plot shows the gating strategy used to define Cisplatin- and Cisplatin+ cells, which were investigated for their signaling responses.

Figure 4B Panels 2-4: Cisplatin+ cells do not signal

Singlets
Cisplatin-
Cisplatin+
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
Singlets Cisplatin- Cisplatin+
Mock stim - 10 mins
  • 0.0
  • 0.0
  • 0.0
125 uM PVO4 - 10 mins
  • 6.81
  • 6.98
  • -0.02

Figure 4B Panels 2-4: Following the gating scheme in Figure 4B Panel 1, induction of pSLP-76/65 was assayed in either all events (Singlets), Cisplatin- events, or Cisplatin+ events. Comparing the Singlets panel to the Cisplatin- panel, the size of the non-responsive peak is greatly diminished when the confounding Cisplatin+ cells are excluded from the analysis.

Figure 5: No DNA damage response in 6hrs

No treatment
Cisplatin 25uM
30min
 
1hr
 
2hr
 
4hr
 
6hr
 
9hr (Etoposide 20uM)
 
Calculated Raw values of Medians using X-Axis channel(s): Aqua, Qdot525-A
No treatment Cisplatin 25uM
30min
  • 222.03
  • 143.29
1hr
  • 180.44
  • 186.52
2hr
  • 242.2
  • 179.43
4hr
  • 185.93
  • 162.16
6hr
  • 158.92
  • 172.29
9hr (Etoposide 20uM)
  • 102.84
  • 758.95

Figure 5: When compared with untreated cells, no induction of phosphorylated Ser139 on H2AX (pH2AX) or cleaved PARP (cPARP) was apparent in KG-1 cells incubated for 30, 60 , 120, 240 and 360 minutes in cisplatin-free media following a 1-minute “pulse” treatment with cisplatin, 25 μM final concentration. A separate aliquot of cells was treated continuously with 20 μM etoposide for 9 hrs as a positive control for the pH2AX and cPARP antibodies.

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