Background

The mitochondria, the powerhouse of the cell, contain a double membrane, termed the outer and inner mitochondrial membranes. Cellular health and function depend largely on mitochondrial function. Mitochondria not only produce energy through oxidative phosphorylation but are also involved in the metabolism of amino acids, lipids, and nucleotides, diverse signaling and redox processes, and quality control and degradation mechanisms, including mitophagy and apoptosis [1]. They can be disrupted in many common diseases, including psychiatric and neurodegenerative disorders, persistent systemic inflammation, cardiac disease, cancer, and diabetes [2]. Oxidative stress and mitochondrial damage have also been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease [3].

The Seahorse 96 XF Analyzer (Agilent Technologies, Santa Clara, CA, USA) has been an effective tool in non-invasively measuring mitochondrial function for the past decade [4]. It is a high-throughput respirometer that is considered the “gold standard” for quantifying mitochondrial function and bioenergetics in cells [5–7]. It measures two key outcomes simultaneously—oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)—and includes assays specifically designed to stress cells and measure metabolic potential (Agilent). The Mitochondrial Stress Test kit, designed by Agilent Technologies, offers three modulators that are introduced into the cell system to produce distinct measurements by shutting down each of the complexes in the electron transport chain in reverse order [8].

Initial addition of oligomycin blocks ATP synthase in the mitochondrial inner membrane and inhibits its function in chemiosmosis. Then, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, is added to the system, selectively removing the regulation of the proton gradient. This allows the maximum respiration rate to be determined. Finally, rotenone and antimycin A are added, which will inhibit complex I and III, respectively, of the electron transport chain [9]. The generated curve for the oxygen consumption rate can be used to obtain various measurements, including basal respiration, ATP turnover, proton leak, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration.

Peripheral blood mononuclear cells (PBMCs) provide selective responses to the immune system and are major cells in the human body’s immunity. They are a heterogeneous cell population consisting of several cell types, including monocytes, T cells, and natural killer cells [10,11]. Assessing OCR and ECAR values in PBMCs has been shown to indicate metabolic stress associated with conditions such as diabetes and neurodegenerative diseases such as Alzheimer’s disease [12–14].

When using PBMCs in Seahorse analysis, it is important to choose an appropriate adhesive compound to immobilize the cells in the Seahorse microplate. Poly-D-Lysine (PDL) is widely used with PBMCs, but limited literature is available on the use of Poly-L-Lysine (PLL). PDL and PLL are the two enantiomers of a synthetic, positively charged polymer called polylysine. Cellular proteases do not degrade PDL, making it the preferred choice in many cases. Both PDL and PLL are useful in promoting cell adhesion to solid substrates and enhancing electrostatic interactions between negatively charged ions of the cell membrane and positively charged surface ions of attachment factors on the culture surface. When adsorbed to the polystyrene surface, poly-D (or L)-lysine increases the number of positively charged sites available for cell binding. Certain cell types prefer this uniform net positive charge, which can subsequently enhance their cell attachment, growth, and differentiation, especially under serum-free and/or low serum conditions. An array of transfected cell lines, glial cells, primary neurons, and neuroblastomas all frequently exhibit improved adhesion and development on surfaces coated with PDL or PLL [15].

Conducting molecular analysis and assays on PBMCs can be challenging, as they are known to be associated with challenges in maintaining cell viability with multiple handling steps throughout the workflow. Many small-scale laboratories are having difficulty finding protocols that allow them to utilize their limited resources to successfully conduct analysis on PBMCs with improved cell viability and feasible consumables. Many small-scale laboratories like ours that conduct Seahorse Flux Analyzer assays using neuronal cell lines utilize PLL as the primary cell coating agent, which is readily available in the laboratory [8,16–18]. Our objective is to compare PDL (primarily recommended for PBMCs in the literature) and our lab’s available PLL to see if it would be a suitable substitute for PDL. If confirmed, additional purchasing of a coating agent could be avoided for laboratories that utilize a wide range of cell types, including PBMCs, in the Seahorse Flux Analyzer. Therefore, in this protocol paper, our primary goal is to present a simple protocol for conducting Mito Stress assay on PBMCs using the Seahorse 96 XF Analyzer, using two commonly available adhesive compounds, PDL and PLL, and compare their effectiveness in the immobilization of freshly isolated PBMCs.