Sorting cells means manipulating them. This induces biological responses of the cells, resulting in functionalities not representing the previous state of the cells, but indicating effects of sorting procedures. Namely in cases that negative selection is not possible, isolated cells are distinct to their previous characteristics. This is true for bead-based sorting or flow cytometric cell separation and heavily skews functional markers of target cells. Of course, this is a limitation for any following investigation of these cells

Bitar, Michael

Sack, Ulrich

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An Eloquent Proof for a Common ChallengeUlrich Sack,* Michael BitarSORTING cells means manipulating them. This inducesbiological responses of the cells, resulting in functionalitiesnot representing the previous state of the cells, but indicatingeffects of sorting procedures. Namely in cases that negativeselection is not possible, isolated cells are distinct to their pre-vious characteristics. This is true for bead-based sorting orflow cytometric cell separation and heavily skews functionalmarkers of target cells. Of course, this is a limitation for anyfollowing investigation of these cells.In this issue (page XXX) in the article entitled “An evalu-ation of T cell functionality after flow cytometry sorting rev-ealed p38 MAPK activation”, Immanuel Andrä andcolleagues combine flow cytometry and streptamer features toanswer a very relevant question: What happens with T cellswhen undergoing flow cytometric sorting, and how can thisactivation be described by easily accessible markers?Sorting cells is a common necessity from research totherapy. From easy technologies such as adhesion to surfaces,physical procedures using density, or protocols making use ofactive cell migration, up to sorting procedures, the approacheshave been refined remarkably and can deliver well-characterized cell populations on demand. Few examples ofnewer approaches are streptamers (1), single cell sortingmethods (2), tracking dye-independent approaches (3), orbead- and column-based technologies (4).The key technology of the research work presented herewas to apply traceless cell sorting by streptamers as a well-established technology (Fig. 1) to sorted cells (1). The idea forthis reversible Fab staining originates from antigen-specificmultimer staining for T cells and was recently alsohighlighted in parts by Cossarizza et al. in 2019 (5). AsMHC–peptide multimers proved to be detrimental for sur-vival of target cells, reversible antigen specific T cell stainingby streptamers was developed by Knabel et al. (6). For thistype of staining, the interaction between monomeric MHCand TCR is so weak that the MHC–peptide complex must bemultimerized for stable binding. Later the idea was widenednot only to make reversible staining available for antigen spe-cific T cells, but also to allow a plethora of surface markers tobe targeted by reversible staining reagents. To achieve thisgoal, antibodies were mutated in their antigen recognition tobe still specific to its antigen, but with low affinity enablingthe spontaneous dissociation from its antigen if monomeric.For this purpose, Fab monomers had to be generated. Afterovercoming the technological challenge of mutating andexpressing of Fab monomers, the already established MHC-Istreptamer system (6) was utilized to multimerize the low-affinity Fab fragments using fluorescence-labeled Streptactinbackbones and thereby allow reversible isolation of virtuallyall cells (1). At low temperatures, the activating/inhibitorystimulus of antibodies and the adverse effects of immunereactions against antibody labeled cells can be minimized.The release of isolation reagents enhances the in vivo func-tionality of regulatory T cells (7). In line with the develop-ment of new reversible staining and cell purification methods,Andrä and colleagues utilized the FAB-TACS®-technology (8)to directly isolate well-defined CD8 positive cell material fromfreshly drawn unlysed venous blood. These cells were used toevaluate MAPK activation by flow cytometry sorting on ahomogenous cell population largely unmodified by prepara-tion effects but highly pure for their readout systems.Intracellular staining of signal transduction moleculesallows today a specific detection of ongoing activation pro-cesses. As shown by Bitar et al. (2019) (9), phosphorylation ofSTAT-5 allows an early, specific, and sensitive detection ofongoing activation and/or proliferation processes. AnInstitute of Clinical Immunology, Medical Faculty, University ofLeipzig, Leipzig, GermanyReceived 6 December 2019; Accepted 11 December 2019*Correspondence to: Ulrich Sack, Institute of Clinical Immunology,Medical Faculty, University of Leipzig, Leipzig, Germany.Email: ulrich.sack@medizin.uni-leipzig.dePublished online 26 December 2019 in Wiley Online Library(wileyonlinelibrary.com)DOI: 10.1002/cyto.a.23958© 2019 The Authors. Cytometry Part A published by Wiley Periodi-cals, Inc. on behalf of International Society for Advancement ofCytometry.This is an open access article under the terms of the CreativeCommons Attribution-NonCommercial License, which permits use,distribution and reproduction in any medium, provided the originalwork is properly cited and is not used for commercial purposes.Cytometry Part A  97A: 168–170, 2020COMMENTARYFigure 1. Schematic drawing of the underlying principles for reversible staining. In the drawing, the square represents the Streptag. Thecloudlet is for the D-Biotin. The pentamer is a backbone (for flow cytometry analysis). The two black beams are the Fab fragmentsoriginating from antibody sequences were point mutated for weaker antigen affinity. Yet by multimerization Fab fragments stably bind totheir antigens presented on cell surfaces. Multimer backbones are fluorescence-labeled for flow cytometry analysis. Fab fragments bindto multimers via Strep-Tag® technology. Addition of D-biotin dissolves multimerization and the weak binding of a monomeric Fabfragment to its antigen leads to the dissociation from the cell surface. Disrupted 0-biotin backbone and Fab fragments can be removed bywashing steps leaving unlabeled cell material. [Color figure can be viewed at wileyonlinelibrary.com]Figure 2. Legend on next page.Cytometry Part A  97A: 168–170, 2020 169COMMENTARYincreasing number of publications using this intracellularstaining method as a surrogate marker indicating ongoingfunctional changes in cells is huge. As an example, the publi-cation by Crawford et al. (2014) (10) should be cited here.This is also true for specific detection of phosphorylated Mapkinase P38, as shown in a typical plot (Fig. 2).Combining both technologies, Andrä et al. here provideevidence that flow cytometric sorting activates cells in a p38dependent matter. This finding should sensitize us forreanalyzing countless publications investigating functionalcharacteristics of sorted cells. Additionally, streptamer tech-nology has been proven another time to be superior in thiscontext. Of course, this is not the only paper addressing thischallenge. In 2014, Beliakova-Bethell et al. (11) investigatedthe effect of cell subset isolation methods on gene expression.They expected that flow cytometric sorting may have animpact on the isolated cells and investigated gene expressionby microarray analysis. Surprisingly, they found only fewalterations in cells sorted by flow cytometry, in contrast topositively selected cells by other methods. They could not finda stress response. Probably, this finding resulted from themicroarray technology applied in this approach, merging thecells for analysis and not combining a functional analysis withphenotypic characterization as done by Andrä et al. in thisvolume. This is also supported by a paper by Weiss et al.(2019) (4) describing activation processes in any kind ofantibody- of even Fab-dependent separation processes. Thiswill also be true for other approaches that apply streptamersto positive cell isolation (12). Here, the technology appliedwas like the streptamer protocol applied by Andrä et al.;remarkably, activation of target cells could be found.The question what the best technology is seems still tobe open. This should be considered in all laboratories sortingcells, independent on the technology applied to. Probably, the100% untouched cell never will be isolated. But the technol-ogy presented here could help understanding this better. Infact, minor changes can result in severe functional alterations,and analyses of the transcriptome may sometimes be not sen-sitive enough.LITERATURE CITED1. Stemberger C, Dreher S, Tschulik C, Piossek C, Bet J, Yamamoto TN,Schiemann M, Neuenhahn M, Martin K, Schlapschy M, et al. Novel serial positiveenrichment technology enables clinical multiparameter cell sorting. PLoS One 2012;7:e35798.2. Zigon ES, Purseglove SM, Toxavidis V, Rice W, Tigges J, Chan LL. A rapid singlecell sorting verification method using plate-based image cytometry. Cytom A 2018;93:1060–1065.3. Elias G, Ogunjimi B, Van Tendeloo V. Tracking dye-independent approach to iden-tify and isolate in vitro expanded T cells. Cytom A 2019;95:1096–1107.4. Weiss R, Gerdes W, Leonhardt F, Berthold R, Sack U, Grahnert A. A comparativestudy of two separation methods to isolate monocytes. Cytom A 2019;95:234–241.5. Cossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S,Agace WW, Aghaeepour N, Akdis M, Allez M, et al. Guidelines for the use of flowcytometry and cell sorting in immunological studies (second edition). Eur JImmunol 2019;49:1457–1973.6. Knabel M, Franz TJ, Schiemann M, Wulf A, Villmow B, Schmidt B, Bernhard H,Wagner H, Busch DH. Reversible MHC multimer staining for functional isolationof T-cell populations and effective adoptive transfer. Nat Med 2002;8:631–637.7. Mohr F, Fischer JC, Nikolaus M, Stemberger C, Dreher S, Verschoor A, Haas T,Poeck H, Busch DH. Minimally manipulated murine regulatory T cells purified byreversible Fab multimers are potent suppressors for adoptive T-cell therapy. Eur JImmunol 2017;47:2153–2162.8. Mohr F, Przibilla S, Leonhardt F, Stemberger C, Dreher S, Müller TR, Fräßle SP,Schmidt GP, Kiene ML, Stadler H, et al. Efficient immunoaffinity chromatographyof lymphocytes directly from whole blood. Sci Rep 2018;8:16731.9. Bitar M, Boldt A, Freitag MT, Gruhn B, Köhl U, Sack U. Evaluating STAT5 phos-phorylation as a mean to assess T cell proliferation. Front Immunol 2019;10:722.10. Crawford TQ, Jalbert E, Ndhlovu LC, Barbour JD. Concomitant evaluation of PMA+ionomycin-induced kinase phosphorylation and cytokine production in T cell sub-sets by flow cytometry. Cytom A 2014;85:268–276.11. Beliakova-Bethell N, Massanella M, White C, Lada S, Du P, Vaida F, Blanco J,Spina CA, Woelk CH. The effect of cell subset isolation method on gene expressionin leukocytes. Cytom A 2014;85:94–104.12. Pelák O, Kužílková D, Thürner D, Kiene ML, Stanar K, Stuchlý J, Vášková M,Starý J, Hrušák O, Stadler H, et al. Lymphocyte enrichment using CD81-targetedimmunoaffinity matrix. Cytom A 2017;91:62–72.FIGURE 2 Detection of phosphorylated p38 in unstimulated (upper panel) and stimulated (lower panel) CD4+ (blue) and CD8+ (red)subsets by flow cytometry. Peripheral EDTA blood was treated with PMA (400 nM for 15 min) or left unstimulated. Blood samples werelysed and fixed by using “lyse and fix” buffer (BD Biosciences) and incubated at 37C in a water bath for 12 min. The cells werecentrifuged, the supernatant was discarded, and the pellet was washed with 4 ml PBS. The samples were permeabilized by using coldperm buffer III (1 ml) (BD biosciences) and left on ice for 30 min. The pellet was washed three times with a fetal bovine serum stain buffer(FBS) (2 ml) (BD Biosciences) and finally resuspended in 200 μl FBS. For flow cytometric analysis, the T cells were stained with PerCP-CyTM 5.5 mouse anti-human CD3 (2.5 μl, clone UCHT1, BD Biosciences), BD phosflow TM Alexafluor 488 mouse anti-humanCD8 (2.5 μl,clone RPA-T8 BD Biosciences), BD phosflow TM PE mouse anti-humanCD4 (5 μl, clone RPA-T4, BD Biosciences) and Alexa Fluor647 mouse anti-human p38 (10 μl, Clone 36/p38, BD Biosciences). After 1 h of incubation in the dark at room temperature, the cells werewashed with 2 ml stain buffer, centrifuged, and were suspended in 300 μl of stain buffer. Mean fluorescence intensity (MFI) of cellspositive for pP38 are shown. [Color figure can be viewed at wileyonlinelibrary.com]170 An Eloquent Proof for a Common ChallengeCOMMENTARY

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