Elsevier

Methods

Volumes 134–135, 1 February 2018, Pages 56-66
Methods

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis

https://doi.org/10.1016/j.ymeth.2017.10.013Get rights and content

Highlights

  • Fluorescent antibodies detect multiple forms of cell death by flow cytomtery.

  • Necroptotic RIP3 up-regulation by TNFα and shikonin.

  • Inhibition of RIP3 up-regulation by necrostatin and RIP1 inhibitor 481.

  • Imagestream analysis of necroptosis, apoptosis and RIP1-dependent apoptosis.

Abstract

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3−ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK’481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

Introduction

The old format of using numbers to classify cell death, Type I being programmed cell death or apoptosis and necroptosis, Type II being autophagy and Type III being necrosis or oncosis had been used for some considerable time and thus the recent re-classification by the NCCD is welcomed [1], [2], [3], [4]. The new classification highlights the ‘state of the art’ and the complexity of cell death processes [5]. The new format uses the terminology of Programmed Cell Death (PCD) to only include developmental and homeostatic apoptosis. While oncosis, the correct name for the process of necrosis (dead cells could have arrived in this state from any of the cell death processes), has been properly included as Accidental Cell Death (ACD). All other forms of cell death include classic caspase dependent apoptosis, RIP1 dependent apoptosis, necroptosis, pyroptosis, ferroptosis, parthanatos and autophagy and have been termed Regulated Cell Death (RCD) [5]. Regulated forms of cell death such as apoptosis and necroptosis are morphologically distinct in that the cell during apoptosis forms blebs on their surface and undergoes DNA fragmentation [1], [2], [3], [6]. While necroptosis involves the swelling of the cell and organelles with consequent rupture of the cell without significant change to the DNA [7].

Biochemically there are numerous pathways involved in both of these forms of cell death, with apoptosis having an intrinsic route initiated by numerous activating agents such as UV-irradiation, TNFα, etoposide and staurosporine. This also involves the depolarization of the mitochondrial inner membrane resulting in dysfunctionality and release of cytochrome c to the cytosol where it binds to Apaf-1 recruiting pro-caspase-9 in an ATP dependent manner to form the apoptosome. This complex activates Caspase-9 which in turn activates effector caspases, which result in fragmentation of the DNA, cell shrinkage, membrane blebbing and the formation of apoptotic bodies [8].

The other route apoptosis can take is the extrinsic route by which TNFα binds to TNFR1 and TNFR2 with signalling via the TRADD death receptor or Fas ligand (Fas L) binding to the FAS receptor. This results in the formation of DISC (Death Inducing Signalling Complex) which contains FADD and caspases 8 and 10 [6], [9]. This complex activates caspase-3 which again leads to cell death via apoptosis (complex IIa) [10]. This activation route can also involve RIP1 (Receptor-interacting serine/threonine-protein kinase 1), RIP3 and caspases resulting in RIP1-dependent apoptosis via complex IIb [6], [10], [11], [12]. Cell death via this cell death receptor route can also bypass the requirement for caspase activation resulting in RIP1 activation and up-regulation of RIP3 resulting in the formation of the so-called ‘necrosome’ or complex IIc with the generation of ROS by mitochondria resulting in cell death via necroptosis [5], [6], [10], [11].

The occurrence and mechanisms of RCD has been in the main confirmed by the use of Western Blot technology. This approach has investigated a particular form of RCD with the use of various activators and inhibitors to determine the signalling pathways involved and the interconnection of numerous forms of RCD e.g. apoptosis, necroptosis and autophagy [13], [14].

Unfortunately only Western Blot and microscopy technology can be used to investigate cell death in an adequate manner. In the main flow cytometry as a technology has been left behind in that currently only apoptosis and associated processes can be measured. These processes include plasma membrane integrity, phosphatidylserine (PS) flipping, mitochondrial dysfunction, Reactive Oxygen Species (ROS) generation, DNA fragmentation and damage [13], [15], [16], [17], [18], [19]. However, none of these flow cytometric techniques actually detects nor measures the numerous specific forms of cell death. Given the knowledge of the proteins involved in signalling pathways and some of their interconnections between the various forms of RCD and the availability of antibodies to such proteins (usually for Western Blotting only) these could be used to semi-quantitate the numerous forms of RCD by flow cytometry. This would perhaps bring flow cytometry to the fore again in studying the interconnections of the numerous forms of RCD in that it may show the presence of immunophenotypically functionally different sub-populations within live and dead cell populations and would shed new light on the mechanisms and the interactivity involved in apoptosis and necroptosis.

Here we describe such an approach by the use of intracellular labelling of RCD signalling proteins, allowing the degree of apoptosis, RIP1-dependent apoptosis and necroptosis in viable and dead cells to be measured flow cytometrically. The processes of apoptosis and necroptosis are known to take several hours to more than 24 h depending on the inducer employed and so the 24 h time point was employed in this study [20], [21], [22], [23]. The assay employs a fixable cell viability dye and anti-active caspase-3 and anti-RIP3 which allows the identification of live cells undergoing apoptosis (Caspase-3+ve/RIP3−ve) and necroptosis (up-regulated RIP3 on RIP3+ve/Caspase-3−ve events) as well as RIP1 dependent apoptosis (RIP3+ve/Caspase-3+ve). RIP3 is always present with RIP1 so it is assumed that such double positive cells are undergoing RIP1-dependent cell death [10]. Equally it is possible to determine the signalling route by which necrotic cells died. So fixable viability dye+ve/Caspase-3+ve cells have died via apoptosis; only RIP3+ve cells have died via necroptosis. Double positive cells for Caspase-3 and RIP3 have died via RIP1-dependent apoptosis.

Here we show that TNFα and the necroptotic drug shikonin can induce multiple forms of cell death in a single culture of a cell line. The use of pan-caspase inhibitor, zVAD and RIP1 inhibitors, GSK’481 or necrostatin-1 followed by treatment with TNFα or necroptotic drug shikonin appeared to show clearly functional phenotypic distributions within sub-populations. Here we show the pathways that are activated in a single population of cells can be induced to undergo necroptosis but also showed high levels of classic and RIP1-dependent apoptosis. This new flow cytometric approach to the study of multiple forms of RCD simultaneously employing low numbers of cells compared to that used in Western Blots opens the possibility of screening leucocytes and other cell types from patient tissues ex vivo.

Section snippets

Induction of necroptosis, RIP1-dependent apoptosis and apoptosis

Jurkat cells (human acute T cell leukaemia cell line) were used as a model to study necroptosis, RIP1-dependent apoptosis and apoptosis. Cells were left untreated or treated with TNFα (100 ng/ml, PeproTech, UK), shikonin for 24 h (0.5 μM, Santa Cruz, USA). Cells were also pre-treated with necroptosis blockers RIP1 inhibitor GSK’481 (GSK, UK, 300 nM) or necrostatin-1 (60 μM Cambridge Bioscience, UK) for 2 h before TNFα or shikonin treatment. Such cells with blocked necroptosis are thus directed

Annexin V and caspase-3 analysis of apoptosis and necroptosis, and RIP1-dependent apoptosis

The use of the caspase-3 mAb on fixed samples showed the limitation of the annexin V assay, in that anti-active caspase-3 appears to be more sensitive than annexin V and generally detects more apoptotic cells (see Supplementary Fig. 1, Fig. 1). This antibody also specifically indicates the mode of cell death either by apoptosis or necroptosis by being ± caspase-3 plus viability marker respectively were annexin V does not specifically indicate the form of cell death (see Supplementary Fig. 1,

Discussion

The use of DNA dyes to detect apoptosis and cell death in the 80’s by the use of cell permeant Hoechst 33342 together with PI required the need for an expensive UV laser line. This was then replaced by the now ubiquitous annexin V flow cytometric assay and has been in use since the mid-90’s [19]. This assay has been extremely useful in the study of apoptosis particularly when combined with cell permeant probes for mitochondrial function, caspase active probes such as FLICA (Fluorescent

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Thank you to Dr. Philip Harris and Dr. Mike Reilly (GSK, Collegeville, PA) for their kind permission to use and publish data relating to RIP1 inhibitor GSK’481. The Imagestream Mk II instrument was purchased by funding from the Wellcome Trust grant 101604/Z/13/Z.

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