Protocol Dictyostelium Cell Fixation: Two Simple Tricks Michael Koonce 1,*, Irina Tikhonenko 1 and Ralph Gräf 2 1 Division of Translational Medicine, Wadsworth Center, NYS Department of Health, Albany, NY 12237, USA; [email protected] 2 Department of Cell Biology, University of Potsdam, 14476 Potsdam-Golm, Germany; [email protected] * Correspondence: [email protected]; Tel.: +1-518-486-1490 Received: 16 June 2020; Accepted: 1 July 2020; Published: 1 July 2020 Abstract: We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system. Keywords: Dictyostelium; cell fixation; microscopy; microtubule; cytoskeleton 1. Introduction The striking mobility of Dictyostelium makes for an interesting and impactful model system to study dynamic cellular events [1]. Directed cell movement, rapid shape changes, and internal reorganization in response to extracellular cues are frequently accentuated here over more sedentary cell types and thus provide novel access to their study and understanding. However, one of the tradeoffs in a dynamic lifestyle is that these cells can be less tightly adherent to their substrate, and thus imaging and downstream structural analyses can present challenges that must be carefully considered. For many years, we have tinkered with fixation and staining conditions in order to image cytoskeletal arrays and GFP-tagged proteins in D. discoideum [2–6]. Likely not alone, we have been challenged with difficulties in getting cells to sufficiently flatten to facilitate imaging and to remain attached to coverslips for downstream structural processing. In this brief report, we would like to share two insights that make a difference in our work, in the hopes that they benefit others as well. 2. Results 2.1. Cell Flattening In the early 19800s, Fukui and colleagues developed an agar overlay procedure for D. discoideum that utilizes thin sheets of agarose to flatten cells and significantly improves some aspects of cellular imaging [7]. This is now a widely used method that gently restricts the experimental depth of field to around 1–3 µm. Cells under agarose can undergo division and remain viable for hours in sealed chambers. This overlay remains our preferred method for live cell imaging, enabling us to follow individual microtubule (MT) motions in a minimal number of focal planes [8,9]. However, the procedure is a bit cumbersome to use for routine cell fixation, especially for multiple coverslips. The agar sheet takes some care to set up, only covers a small portion of the coverslip, and takes slightly longer for the fixative to penetrate to the cells, and cell loss (at least in our hands) can be significant upon sheet removal for downstream cell processing. With any perturbation, there are caveats to consider when flattening cells for long periods, and thus multiple strategies can be useful to distinguish between genuine and imposed phenotypes. Methods Protoc. 2020, 3, 47; doi:10.3390/mps3030047 www.mdpi.com/journal/mps Methods and Protoc. 2020, 3, 47 2 of 6 As such an alternative, we have begun to spin cells directly onto coverslips. Five minutes at 400 g in a standard tabletop centrifuge not only promotes attachment but also cell spreading. A small volume of cell suspension is added to buffer overlying a coverslip in a centrifuge tube (Figure 1) and spun for five minutes. The coverslip is then lifted out and gently slid into a dish containing fixative. This approach eliminates the waiting period for cell settling; agarose is not required, and cells labeled with a tubulin antibody [10] appear very similar to those prepared by agar overlay (Figure 2). Methods Protoc. 2020, 3, 47 2 of 6 The take-home message here is that the organization of the MT array in centrifuged cells appears to be very similar if not identical to what we would select as the “best” cells from either direct fix or agar Asoverlay such procedures. an alternative, However, we have a primary begun to benefit spin cells is that directly they are onto significantly coverslips. more Five plentiful minutes on at 400the centrifuged g in a standard coverslip tabletop than centrifuge in the other not onlytwo pr promoteseparations. attachment For example, but also approximately cell spreading. 10% A of small the volumecells on ofa gravity cell suspension settled coverslip is added are to busufficientlyffer overlying flat enough a coverslip to project in a centrifuge a useful tubecharacterization (Figure1) and of spunMT arrays for five for minutes. the types The questions coverslip that is interest then lifted us. On out a andcentrifuged gently slid coverslip, into a dish this containingincreases to fixative. at least This60% approachof the population. eliminates This the percentage waiting period is similar for cell in settling; the agar agarose overlay is notpreparations, required, andbut cellscentrifuged labeled withcells aextend tubulin across antibody the entire [10] appearcoverslip very and similar do no tot suffer those detachment prepared by due agar to overlay agar sheet (Figure removal.2). Figure 1. CentrifugationCentrifugation devices. devices. Panel Panel (A ()A shows) shows a side a side view, view, panel panel (B) (showsB) shows a top a topdown down view view of a of50 amL 50 Sorvall mL Sorvall centrifugation centrifugation tube fitted tube fittedwith an with EPON an EPON base (yellow base (yellow layer in layer A) and in A a) white and a Delrin white Delrinrod segment rod segment (2.5 cm (2.5 diameter, cm diameter, 3.75 3.75cm cmlength). length). The The notch notch along along the the side side is is helpful helpful to to retrieve coverslips withwith forceps,forceps, allowingallowing accessaccess to to the the underside underside of of the the coverslip coverslip for for lifting. lifting. Panel Panel (C ()C shows) shows a modifieda modified multiwell multiwell holder holder for for processing processing up up to to 12 12 samples samples at at a a time. time. TheThe toptop rowrow containscontains 1212 mm round coverslips;coverslips; thesethese cancan bebe readilyreadily removedremoved withwith curvedcurved forceps.forceps. The take-home message here is that the organization of the MT array in centrifuged cells appears to be very similar if not identical to what we would select as the “best” cells from either direct fix or agar overlay procedures. However, a primary benefit is that they are significantly more plentiful on the centrifuged coverslip than in the other two preparations. For example, approximately 10% of the cells on a gravity settled coverslip are sufficiently flat enough to project a useful characterization of MT arrays for the types questions that interest us. On a centrifuged coverslip, this increases to at least 60% of the population. This percentage is similar in the agar overlay preparations, but centrifuged cells extend across the entire coverslip and do not suffer detachment due to agar sheet removal. Methods Protoc. 2020, 3, 47 3 of 6 Methods and Protoc. 2020, 3, 47 3 of 6 FigureFigure 2. ExamplesExamples of of antibody-labeled antibody-labeled microtube microtube (MT) (MT) patterns patterns in cells in (A cells) directly (A) addeddirectly to added coverslips to coverslipsand fixed afterand afixed 10 min after period; a 10 min (B) cellsperiod; fixed (B after) cells overlay fixed withafter agaroverlay sheets; with and agar (C ,sheets;D) cells and fixed (C after,D) cells5 min fixed centrifugation after 5 min onto centrifugation coverslips. onto Panels coverslips. (A–C) show Panels interphase (A)–(C)arrangements show interphase of mononucleated arrangements ofcells mononucleated (left) and binucleates cells (left) (right). and Panelbinucleates (D) shows (right). mitotic Panel cells (D) in shows late anaphase mitotic (left)cells andin late late anaphase telophase (left)(right). and While late telophase the three (right). different While cell the preparations three differe looknt cell similar, preparations it is important look similar, to note it that is important there are totypically note that far there fewer are flattened typically cells far on fewer the direct flattened fix coverslips cells on the and direct substantially fix coverslips fewer and cells substantially in general on fewerthe agarose cells in overlay general coverslip. on the agarose MTs are overlay shown cove in green,rslip. MTs DNA are in blue,shown Scale in green, bar = 5DNAµm. in blue, Scale bar = 5 μm. 2.2. Cell Attachment 2.2. CellThe Attachment second perhaps underappreciated impact derives from a fixation recipe that includes a smallThe amount second of perhaps glutaraldehyde underappreciated (0.05% final). impact This derives concentration from a fixation is not recipe sufficient that includes to preserve a small cell amountstructure of on glutaraldehyde its own, nor does (0.05% it require final). This autofluorescence concentration quenching is not sufficient by sodium to preserve borohydride, cell structure but it substantially improves cell retention on the coverslip (Figure3). Although formaldehyde alone or 20 on its own, nor does it require autofluorescence quenching by sodium borohydride, but− it◦ substantiallymethanol can improves provide structuralcell retention preservation on the coverslip at the light (Figure microscopy 3). Although (LM) formaldehyde level, cell loss alone from theor −coverslip20° methanol is substantial can provide at each structural wash or preservation incubation step. at the This light is particularlymicroscopy a(LM) problem level, for cell mitotic loss from cells, thesince coverslip they are is more substantial rounded at each and lesswash adherent or incubation than during step. This interphase. is particularly The fixation a problem recipe for provided mitotic cells,below since does they not interfereare more with rounded GFP labels, and less and adherent thus expressed than during tags can interphase.