TITLE: Protocol for high-throughput isolation of bacterial intracellular nonreplicating persisters
AUTHORS: Iris Dadole, Kevin Huguet, Didier Blaha, Nicolas Personnic

[DESCRIPTION]
Failure of antibiotics to clear a bacterial infection is arguably one of the most severe threat to global health. Bacteria ability to withstand drug treatment partly relies on the formation of a subpopulation of persisters. Persisters are rare nonreplicating individuals, transiently refractory to antibiotic treatment and responsible for non-resolving bacterial infection and relapse. Studying persisters is challenging as it is a reversible physiological state occurring in a small fraction of the pathogen population hidden in a complex environment. Persisters have been primarily analyzed in broth, hardly reflecting the conditions prevailing within a host. In addition, indirect read-outs used to detect the persisters, a posteriori, limited a comprehensive understanding of the persisters peculiar physiology during the infection. Here, we describe a high-throughput method to track, and collect the bacterial persisters generated during the infection of phagocytic cells, with single-cell resolution. Persisters are detected based on the bacterial division rate measured for each individual bacterium using the Timer fluorescent growth rate reporter and subsequently recover using fluorescence-activated cell sorting (FACS). We will illustrate this method with Legionella pneumophila, a facultative intracellular pathogen and the agent of the Legionnaires’ disease for which persistence to antibiotics and relapsing infections are clinically documented. Isolated persisters can be analyzed biochemically and functionally, yielding fundamental new insights into the molecular and cellular processes underlying persistence, and into the peculiar physiology of the persisters within the host. This method is adaptable to other microbial pathogens and alternative infection models.

[GUIDELINES]
SUMMARY:

Persisters represent a subpopulation of nonreplicating individuals transiently refractory to antibiotic treatment. Here we describe a high-throughput single-cell method to track and collect the persisters formed within phagocytes. Cells are infected with bacteria expressing the Timer fluorescent growth rate reporter, and nonreplicating individuals are recovered using fluorescence-activated cell sorting (FACS).

INTRODUCTION: 

During the colonization of the host, the pathogen encounters highly heterogeneous host micro-environments [1], elaborate a complex interplay with the component of the immune systems [2] and/or develops microbial communities with high degree of phenotypic specialization [3] which produce, within the micrometer ranges, a multitude of genetically identical individuals with alternative physiologies [4]. Bacterial variability is further increased as clonal bacteria constantly develop functionally distinct subpopulations [5]. This reversible and regulated phenomenon termed phenotypic heterogeneity is of major clinical importance as it implies the formation of persisters [6]. Persisters are preestablished or stress induced individuals that transiently evade the bactericidal activity of antibiotics by entering a rare and peculiar physiological state at the cost of abrogation of bacterial division, and finally resume growth on the onset of the treatment termination [7]. The emergence of persisters has been documented for major bacterial pathogens including Staphylococcus aureus, Mycobacterium tuberculosis, Escherichia coli, Salmonella enterica, Pseudomonas spp., Listeria monocytogenes and Legionella pneumophila [4,6,8–16]. Traditionally, the presence of bacterial persisters has been revealed by the “biphasic killing” phenomenon of bacterial cultures exposed to bactericidal antibiotics [17]. However, such population-averaging methods, performed a posteriori, are poorly suited to comprehensively and dynamically analyze a phenomenon where reversible cell-to-cell variations plays a key role. The development of high-throughput fluorescence-based single-cell technologies has rendered important breakthroughs reachable. Notably, the microfluidic device called mother machine has been incredibly useful to directly monitor the formation of persisters and the underlying regulatory mechanisms using a combination of genetic and environmental manipulations [6]. Persisters have been mostly studied using non-infectious model organisms cultivated in broth, hardly reflecting the conditions prevailing within a host. Deciphering the biology of the persisters during the infection has remained technically challenging as the persisters represent a rare and unstable subpopulation of the pathogen, that is generated within complex environments. 

The ubiquitous environmental Gram-negative bacterium Legionella pneumophila is a waterborne human pathogen responsible for a life-threatening pneumonia termed Legionnaires’ disease (LD) [18], the inventoried cases of which have doubled since 2013 [19]. LD is fatal for 5-15% of the patients (up to 50% among the elderly), even when promptly diagnosed and treated [20]. A nation-wide retrospective study describing those cases of slowly or non-resolving LD proposed that recalcitrance to antibiotic treatments or relapsing infections were the expression of bacterial persistence [21]. L. pneumophila survives its ingestion by a diverse array of protozoan predators, as well as lung alveolar macrophages, and establishes a replicative membrane-bound compartment termed the Legionella-containing vacuole (LCV) [22,23]. LCV formation requires the Icm/Dot type IV secretion system (T4SS) and involves approximately 300 different so-called « effector » proteins, which are translocated into host cells [24,25] to hijack numerous host cell functions. The amoebozoa Acanthamoeba castellanii has proved a reliable, easy-to-use and cost effective model organism to investigate the molecular interactions of L. pneumophila with its host cell, and at large, the pathogenic potential and process of many other human opportunistic pathogens [26]. 

The plasmid based Timer growth rate reporter [4] is a powerful technology to track the nonreplicating persisters produced during an infection. Timer is a stable fluorescent protein that slowly maturates from a green to a red fluorescent protein. As a consequence, in growing bacteria, constitutively producing Timer, green fluorescent Timer dominates over red fluorescent Timer, which is diluted by cell division before maturation, and the individual bacteria show a high [500 nm (green)/600 nm (red)] fluorescence (color) ratio (i.e. “green” fluorescence). By contrast, growth-arrested bacteria accumulate both green and red fluorescent Timer, and the individual bacteria show a low green/red color ratio (i.e., “red” fluorescence).

Here, coupling the protozoan model of infection, the innovative Timer fluorescent growth rate reporter and high-throughput single-cell technologies, we are defining a pipeline to track and collect the persisters produced by L. pneumophila with an unprecedented resolution. Isolated persisters can be analyzed biochemically and functionally. To date, using this methodology, we demonstrated that L. pneumophila persisters are a preestablished subpopulation of nongrowers and the expression of a regulated pathogen strategy which is applied to evolutionarily distant host cells (i.e., protists and macrophages) [15,16]. Near in situ biochemical characterization of the intracellular L. pneumophila persisters unveiled a unique physiology and, remarkably, a specific virulence program in order to survive the combined action of the host defenses and the antibiotics, hence, breaking the dogma that persisters are dormant bacteria [15]. Of note, this approach can be applied to the study nonreplicating persisters produced by other pathogens during the infection or within complex microbial communities.

ACKNOWLEDGMENTS:

Research at the CIRI is supported by the Inserm, CNRS, University Lyon 1, and ENS Lyon. N.P. was the recipient of an SNF Ambizione fellowship (PZ00P3_161492, PZ00P3_185529). The research in the laboratory of N.P. is supported by a CIRI seed fund and the ANR. The funders had no role in study design, decision to publish, or preparation of the manuscript.

DISCLOSURES:

The authors have nothing to disclose.

REFERENCES:

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[STEPS]
SECTION: Cultivation of Acanthamoeba castellanii: Axenic cultivation of A. castellanii trophozoites
1. 
 
Prepare Peptone Yeast Glucose (PYG) broth (see Note 3): 
 
      yeast extract  1 g/L BBL    Bacto Proteose Peptone  20 g/L    D(+)glucose monohydrate (50 mL of a 2 M  1.8% (w/v)    MgSO4 (10 mL of a 0.4 M solution)  4 mM    CaCl2 (8 mL of a 0.05 M solution)  0.4 mM    sodium citrate (3.4 mL of a 1 M solution)  3.4 mM    Fe(NH4)2(SO4)2 (20 mg)  0.05 mM    Na2HPO4 (10 mL of a 0.25 M solution)  2.5 mM    KH2PO4 (10 mL of a 0.25 M solution)   2.5 mM  
 
Add the components (except the glucose) to 950 mL H2O, adjust the pH with 1 Molarity (M) HCl to 6.5±0.1 and autoclave. Solubilize 11 g of D(+)glucose in 50 mL of warmed H2O (37 °C) and add to the autoclaved medium (see Note 4). Sterilize the medium using a   filter cartouche and store at 4 °C.
 

SECTION: Cultivation of Acanthamoeba castellanii: Axenic cultivation of A. castellanii trophozoites
2. Thaw an ampule of frozen amoeba at Room temperature and aseptically transfer the contents into a 13 mL sterile Falcon round-bottom polypropylene tubes containing 10 mL of PYG. Centrifuge 250 x g, 10 min and discard the supernatant. Resuspend the pellet in 10 mL of PYG medium to inoculate a tissue culture treated flask (75 cm2: 10-15mL medium) for initial amplification.

SECTION: Cultivation of Acanthamoeba castellanii: Axenic cultivation of A. castellanii trophozoites
3. Cultivate the amoebae at 23 °C in PYG medium optionally supplemented with antibiotics (e.g. Pen/Strep or Fungizone) to avoid bacterial and fungal contaminations. Use either 100 mm tissue culture treated Petri dishes (minimum of 10 mL medium) or tissue culture treated flasks (25 cm2: 5-7 mL medium, 75 cm2: 10-15mL medium). Change the medium every 2-3 days. Split the culture when cell reach 80% confluence by repeated pipetting of the media over the plate. 

SECTION: Cultivation of Acanthamoeba castellanii: Cryopreservation of A. castellanii
4. Remove the PYG medium and harvest the amoeba, when in logarithmic growth phase, by repeated pipetting of fresh refrigerated PYG medium over the plate. Determine the cell concentration by counting the amoeba with a haemocytometer. Adjust the concentration to about 107 amoeba/mL.
SECTION: Cultivation of Acanthamoeba castellanii: Cryopreservation of A. castellanii
5. Prepare freshly a 15% solution of sterile dimethyl sulfoxide (DMSO) in refrigerated PYG medium and mix to the cell suspension in equal portions to reach a final concentration between 106 and 107 cells/mL and 7.5 % DMSO.
SECTION: Cultivation of Acanthamoeba castellanii: Cryopreservation of A. castellanii
6. Dispense in 0.5 mL aliquots into 2.0 mL sterile plastic screw-capped CryoTube vials. Place the vials in a Nalgene Cryo 1 °C freezing container and store at -80 °C,  .
 


SECTION: Generation of a Timer producing Legionella pneumophila
7. 
 
Prepare CYE (charcoal yeast extract) agar plates: 
 
      ACES  10 g/L    Bacto™ yeast extract (see Note 7)  10 g/L    activated charcoal powder (puriss. p.a.)  2 g/L    agar  15 g/L    L-cysteine  3.3 mM    Fe(NO3)3  0.6 mM  
 
Dissolve 10 g of ACES and 10 g of yeast extract in 950 mL of H2O and adjust the pH to pH 6.9 with 10 Molarity (M) KOH. Transfer the solution to a 1 L Schott bottle containing 2 g of activated charcoal powder, 15 g of agar and a stir bar. Autoclave and let the agar solution cool down to 50 °C. Add filter sterilized 0.4 g/10 mL L-cysteine and 0.25 g/10 mL Fe(NO3)3 solutions (see Note 8). If required add antibiotics. Mix the solution on a magnetic stirrer and pour plates (approximately 40 plates per L of medium). Let the plates dry for 1 day at Room temperature and store at 4 °C.
 

SECTION: Generation of a Timer producing Legionella pneumophila
8. Prepare ACES yeast extract (AYE) broth: 
 
      N-(2-acetamido)-2-aminoethane-sulfonic   acid (ACES)  10 g/L    Bacto yeast extract  10 g/L    L-cysteine  3.3 mM    Fe(NO3)3  0.6 mM  
 
Add 10 g of ACES and 10 g of yeast extract in 950 mL of H2O. Add filter sterilized 0.4 g/10 mL L-cysteine and 0.25 g/10 mL Fe(NO3)3solutions. Adjust the pH to pH 6.9 with 10 Molarity (M) KOH. If required, add antibiotics. Pass the medium several times through a glass-fibers filters, and finally sterilize the medium using a   filter cartouche. Store the medium at 4 °C in the dark (see Note 9).
 

SECTION: Generation of a Timer producing Legionella pneumophila: Preparation of electrocompetent L. pneumophila
9. 
 
Streak out L. pneumophila wild-type and the isogenic mutant DicmT from frozen glycerol stocks onto CYE plates. Colonies or a bacterial lawn will form after 3 days of incubation at 30 °C (see Note 11).
 


SECTION: Generation of a Timer producing Legionella pneumophila: Preparation of electrocompetent L. pneumophila
10. Generously harvest the bacterial lawn with a disposable inoculation loop and inoculate 1 mL of PBS in a 1.5 mL sterile Eppendorf tube.

SECTION: Generation of a Timer producing Legionella pneumophila: Preparation of electrocompetent L. pneumophila
11. Centrifuge at 5000 rpm, 10 min, discard the supernatant and resuspend the bacterial pellet with a 10 % glycerol solution. Repeat the operation 3-4 times.
SECTION: Generation of a Timer producing Legionella pneumophila: Preparation of electrocompetent L. pneumophila
12. Finally, resuspend the bacterial pellet with 100 mL of a 10% glycerol solution.

SECTION: Generation of a Timer producing Legionella pneumophila: Preparation of electrocompetent L. pneumophila
13. Directly proceed with the electroporation. 
SECTION: Generation of a Timer producing Legionella pneumophila: Electroporation of L. pneumophila with Timer growth rate reporter
14. Add 100 ng of the plasmid pNP107 to the bacterial suspension and transfer into a 2 mm electroporation cuvette.

SECTION: Generation of a Timer producing Legionella pneumophila: Electroporation of L. pneumophila with Timer growth rate reporter
15. Electroporate using the following settings: U=2.5 kV; C= 25 µF; 200 Ω=∞; t.c.≈2.5.
SECTION: Generation of a Timer producing Legionella pneumophila: Electroporation of L. pneumophila with Timer growth rate reporter
16. Immediately add 500 µL of pre-warmed AYE, transfer into a 13 mL Falcon round-bottom polypropylene tubes and incubate on a shaker set at  .

SECTION: Generation of a Timer producing Legionella pneumophila: Electroporation of L. pneumophila with Timer growth rate reporter
17. Plate on CYE agar supplemented with 5 µg/mL chloramphenicol (see Note 12). Orange colonies (i.e.,producing the Timer fluorescent protein) will form after 3 days of incubation at 37 °C.
 

SECTION: Preparation of infectious Legionella pneumophila
18. Streak out Timer producing L. pneumophila from frozen glycerol stocks onto CYE plates supplemented with 5 µg/mL chloramphenicol. Colonies or a bacterial lawn will form after 2-3 days of incubation at 37 °C.
SECTION: Preparation of infectious Legionella pneumophila
19. Prepare planktonic cultures for infection (see Note 13). Suspend a loop of the bacterial lawn grown on CYE/Cam agar plates for 2-3 days in 600 µL of AYE medium supplemented with 5 µg/mL chloramphenicol (AYE/Cam) in a 2 mL Eppendorf tube. Mix thoroughly until you reach a homogeneous suspension (see Note 14). Measure the OD600 nm and adjust the density of the bacterial suspension to an OD600 nm of 0.1. Inoculate 9 mL of AYE/Cam with the suspension to obtain an OD600 nm of 0.1 and split into 3 x 3 mL in 13 mL FalconTM round-bottom polypropylene tubes and incubate on a shaker set at  .
 

SECTION: Preparation of infectious Legionella pneumophila
20. At 21 h, L. pneumophila must be transmissive. Check the bacteria by examining 10 µL of culture loaded on a glass slide covered with a 12 mm round glass coverslip with an inverted light microscope (40 × objective). L. pneumophila look like a dense population of small and motile coccobacilli (see Note 15). Measure the OD600 nm; an OD600 nm of 5 corresponds to approximately 2 × 109 bacteria/mL (see Note 16).
 

SECTION: Preparation of infectious Legionella pneumophila
21. Proceed with infection of A. castellanii.
SECTION: Infection of Acanthamobea castellanii
22. Split A. castellanii cultures one day before the experiment to obtain cells in logarithmic growth phase, which have been fed the day before (see Note 17).
 

SECTION: Infection of Acanthamobea castellanii
23. Wash exponentially growing A. castellanii with PYG and detach the cells by repeated pipetting of the media over the plate.
SECTION: Infection of Acanthamobea castellanii
24. Determine cell concentration by counting the cells with a haemocytometer.
SECTION: Infection of Acanthamobea castellanii
25. Seed cells at a density of 9 x 105 amoeba per well of a tissue culture treated 6-well plate. Let the phagocytes settle and adhere to the plastic surface for 1440 min under normal culture conditions (see Note 18).
 


SECTION: Infection of Acanthamobea castellanii
26. Prior to infection, remove the PYG medium and wash out antibiotics if necessary (see Note 19). Determine the cell numbers per well using a haemocytometer. Calculate the appropriate number of bacteria to infect host cells with a multiplicity of infection (MOI) of 2.
 

SECTION: Infection of Acanthamobea castellanii
27. Synchronize the infection by centrifugation of the 6-well plate for 250 x g, 10 min and incubate for 45 to 60 min at 25 °C. Remove not internalized bacteria by washing.
Wash the infected cells with PYG. (1/3)
Wash the infected cells with PYG. (1/3)
Wash the infected cells with PYG. (1/3)
Incubate at least 1440 min at 25 °C (see Note 20 & 21).
 

SECTION: Infection of Acanthamobea castellanii
28. Proceed with the sample preparation for FACS.
SECTION: Sample preparation for FACS
29. 
 
Prepare homogenization (HS) buffer:  
      N-2-hydroxyethylpiperazine-N-2-ethanesulfonic   acid (HEPES)  20 mM    sucrose  250 mM    ethyleneglycoltetraacetic acid (EGTA)  0.5 mM    pH adjusted to 7.2 with KOH  1 M  

SECTION: Sample preparation for FACS
30. on ice, detach the infected amoeba by repeated pipetting of the media over the wells and transfer into 2 mL Eppendorf tubes.
SECTION: Sample preparation for FACS
31. Pellet by centrifugation 250 x g, 10 min, 4 °C, remove the supernatant and wash once with 1 mL ice-cold PBS.
SECTION: Sample preparation for FACS
32. on ice, lyse the cell pellet with 1 mL 0.1 % Triton TX-100 (Sigma) in HS buffer for 10 min.
SECTION: Sample preparation for FACS
33. Centrifuge the cell lysate 250 x g, 15 min, 4 °C, remove the supernatant and resuspend the pellet with 1 mL PBS. Keep on ice.

SECTION: Sample preparation for FACS
34. Assemble and place the Isobiotech ball homogenizer (http://www.isobiotec.com) (6 or 8 mm clearance ball) on ice. Mount a pair of Luer-lock syringes.

SECTION: Sample preparation for FACS
35. Rinse well the ball homogenizer. 
SECTION: Sample preparation for FACS
36. Install a new pair of Luer-Lock syringes. Remove the plunger of one syringe to load the cell lysate. Install the plunger and press the sample through the homogenizer into the second syringe. Press back and forth nine times (see Note 24).
 

SECTION: Sample preparation for FACS
37. Collect the homogenized sample and transfer trough the cell-strainer cap into a 5 mL polystyrene round-bottom tube (see Note 25). Keep on ice.
 

SECTION: Sample preparation for FACS
38. Proceed with the isolation of the nonreplicating persisters.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
39. 
 
Start up the sorter.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.1. Decontaminate the sort block, the sort chamber, the sample injection port and the sort collection device (tube holder and lid) with 70% ethanol. Clean the deflecting plates with ddH2O. Wipe to dry carefully all surfaces that will be charged.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.2. Assemble and filled the sheath tank with sterile PBS (see Note 28 & 29).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.3. Turn on the aerosol management system.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.4. Turn on the sorter (see Note 30).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.5. In the FACSDiva menu, turn on the fluidic system by selecting “Fluidic startup”(see Note 31) and follow the instructions.
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.6. In the sort block, remove the close-loop nozzle and insert the nozzle with an orifice of 70 mm at the dedicated position at the lower end of the cuvette flow cell. Set the correct configuration for the 70 mm nozzle in the FACSDiva menu “view configurations”.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
39.7. Turn on the external temperature control system for the collection tube holder and set the temperature to 4 °C. In the FACSDiva menu “Cytometer”, set the agitation of the sample in the injection chamber to   and temperature to 4 °C.

SECTION: High-throughput isolation of nonreplicating persisters by FACS
40. Establish a stable stream and set up the drop formation. 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.1. Start the stream that should appear in the breakoff window.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.2. Within the sort block, ensure the stream hit the center of the waste aspirator (see Note 34).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.3. Stabilize the position of the drop breakoff point by adjusting the amplitude (see Note 35). Drops before the breakoff point will appear pear-shaped. Ensure satellite drop merge with leading drop.
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.4. Copy the generated Drop 1 value (i.e., the distance between the top of the image and the center of the first broken-off drop) in the target field. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.5. Turn on the Sweet Spot to maintain the drop breakoff point during the sorting.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
40.6. Wait 15-20 min and control the stability of the stream and drop formation. For the Drop 1, a difference of 10 units is acceptable. For the GAP a difference of 1 unit is acceptable.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41. Adjust the drop delay.
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.1. Ensure the maximal illumination of the central stream using the micrometer dial.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.2. Turn on the deflection plates (voltage) in the side stream window (see Note 39). 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.3. In the FACSDiva menu, create an AccuDrop experiment.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.4. Place a tube filled with a dilute suspension of AccuDrop beads on the sample injection port and load into the sample injection chamber (see Note 40). Adjust the flow rate to reach a threshold rate of 2’000-3’000 events per second (see Note 41).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.5. Activate the test sort in the side stream window. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.6. Activate the optical Filter in the side stream window.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.7. Adjust the voltage slider to place the left side stream in the center of the box on the left. Ensure that the central stream is visible in the right box (see Note 42).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.8. Stop the test sort in the side stream window.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.9. In the FACSDiva sort layout window select “2 tube” as device and “Initial” as a precision mode. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.10. Start sorting the beads by selecting “sort” in the sort layout window (see Note 43).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.11. In the side stream window, adjust the drop delay value in 1-drop increments to achieve close to 100% intensity in the left side stream (see Note 44).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.12. In the sort layout window, change the precision mode to “Fine Tune” and continue optimizing the drop delay by small increments until the left side stream intensity is > 95 % (see Note 45).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.13. In the side stream window, inactivate the Optical Filter and set the voltage slider to “0”. In the sort layout, stop the sort. Close the AccuDrop experiment. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
41.14. Unload the tube filled with AccuDrop beads using the FACSDiva acquisition dash board. Discard the tube or keep it in a fridge for future sort.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
42. Set up the side streams for the 2-way sort.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
42.1. Insert one 15 mL tube in the 2-way sort tube holder. Close with the universal top. Insert the sort collection device into the sort collection chamber and connect to the temperature control system in order to refrigerate the collection tube.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
42.2. In the side stream window, turn on the deflection plates (voltage) and activate “test sort”. Optimize the position of the first either right or left side stream using the corresponding voltage sliders (see Note 46).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
42.3. In the side stream window, open the aspirator drawer to confirm the side stream are properly aligned with the collection 15 mL tube.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
42.4. Close the aspirator drawer, inactivate “test sort”, turn off the voltage, remove the sort collection device and trash the 15 mL collection tube. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43. Create the gates to sort nonreplicating persisters. 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.1. In FACSDiva browser, open a new experiment and click on “Cytometer settings”. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.2. In the Cytometer window, in the parameter tab, delete all unneeded optical configuration. Keep forward scatter (FSC); side scatter(SSC); [Laser 488 nm - 502LP - BP 530/30] (FITC) and [Laser 561 nm - 600LP - BP 610/20] (mCherry).
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.3. For each parameter, only record the height (H) of the pulse signal detected.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.4. In the cytometer window, in the threshold tab, set both the FSC and SSC values to the minimum (i.e., 200).
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.5. On the global worksheet, create (i) a SSC-H vs FSC-H plot; (ii) a FITC-H vs FSC-H plot and (iii) a mCherry-H vs FITC-H plot. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.6. In the inspector window, select the checkbox for X Axis and Y Axis under biexponential display.

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.7. Place the sample tube filled with homogenized lysates on the sample injection port and load into the sample injection chamber using the FACSDiva acquisition dash board and start acquisition (see Note 49). Detected signals appear in the plots created on the global worksheet.
 


SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.8. In the cytometer window, parameter tab, set the photomultiplier (PMT) voltage for FSC and SSC in order to center the detected signal on the SSC-H vs FSC-H plot.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.9. In the cytometer window, parameter tab, set the PMT voltage for FITC-H in order to separate the green Timer signal from the host debris autofluorescence and the electronic noise on the FITC-H vs FSC-H plot (see Note 50). Using the polygonal gating tools, draw the Gate 1 around the FITC-H positive population. 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.10. In the mCherry-H vs FITC-H plot, apply Gate 1. In the cytometer window, parameter tab, set the PMT voltage for mCherry-H in order to center the red Timer signal (see Note 51). 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.11. In the mCherry-H vs FITC-H plot, two well-separated subpopulations with similar FITC-H signal intensity appear. The population with the higher mCherry intensity corresponds to the nonreplicating persisters. Using the gating tools, draw the Gate 2 around this subpopulation (see Note 52 and 53). 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.12. In the FACSDiva acquisition dash board, select Gate 2 as stopping gate and 10’000 as events to record. Start recording by clicking “record” (see Note 54).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
43.13. In the mCherry-H vs FITC-H plot, using the population hierarchy view, determine the population size in Gate 2 (% Parent).
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44. Sort the nonreplicating persisters. 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.1. Insert one 15 mL tube prefilled with 3 mL ice-cold PBS in the 2-way sort tube holder. In the sort layout window, define the sort location field to align the collection tube with the side stream as setup in 5.3. In the selected sort location field, add the subpopulation to be sorted (i.e., Gate 2). Close with the universal top. Insert the sort collection device into the sort collection chamber (as in 42.1.).

SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.2. In the FACSDiva sort layout window select “2 tube” as device and “purity” as a precision mode (see Note 56). 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.3. In the side stream window, turn on the deflection plates (voltage), open the aspirator drawer and initiate the sample flow (“acquire”). 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.4. In the FACSDiva sort layout window, lunch the sort by clicking “sort”.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.5. In the sort layout window, monitor the sorting by observing the sorting rate, the conflicts rate and the sorting efficiency (ideally above 95 %). Inspect regularly the central and side stream in the side stream window. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.6. Sort until the required number of nonreplicating persisters has been sorted (see Note 57-58).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.7. Stop the sort by inactivating “sort” in the sort layout window and save the “sort report”. In the side stream window, control the deflection plates (voltage) are turned off and the aspirator drawer closed.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.8. Unload the sample tube using the FACSDiva acquisition dash board and eliminate into the dedicated biological wastes disposal in compliance with local rules.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
44.9. Open the sort chamber, remove and disassemble the sort collection device to collect and the store on ice the 15 mL collection tube.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45. Re-analysis of the sorted nonreplicating persisters. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.1. In the FACSDiva browser, under experiment and specimen create a new tube (see Note 59).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.2. Harvest only 100 mL of sorted nonreplicating persisters from the 15 mL collection tube and transfer into a sample tube. 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.3. Place the sample tube on the sample injection port and load into the sample injection chamber using the FACSDiva acquisition dash board and activate acquisition. Detected signals appear in the plots created on the global worksheet.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.4. In the FACSDiva acquisition dash board, adjust the flow rate to reach a threshold rate of 2’000-3’000 events per second.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.5. In the FACSDiva acquisition dash board, select Gate 2 as stopping. Start recording by clicking “record”. Stop recording and acquisition after 500-1000 events are recorded (see Note 60).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.6. In the mCherry-H vs FITC-H plot, using the population hierarchy view, determinethe population size in Gate 2 (% Parent) to infer the sort purity.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.7. Unload the sample tube using the FACSDiva acquisition dash board and eliminate into the dedicated biological wastes disposal in compliance with local rules.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
45.8. Save your date and proceed with the shut-down. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46. Shut-down. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.1. Clean by sequentially loading and running sample tubes filled with FACSClean FACSRinse, 70% ethanol and finally sterile ddH2O.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.2. In the side stream window, turn off the stream, depressurized and disassemble the PBS tank (see Note 61). 
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.3. Decontaminate the sort block, the sort chamber, the sample injection port, the sort collection device (tube holder and lid) with 70% ethanol. Clean the deflecting plates with ethanol and ddH2O. Wipe to dry carefully all surfaces.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.4. Assemble and filled the ethanol tank with 70% ethanol and connect to the sorter fluidics system (see Note 62).
 

SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.5. In the FACSDiva menu, turn off the fluidic system by selecting “Fluidic shutdown” and follow the instructions.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.6. In the sort block, remove the 70 mm nozzle. Decontaminate the nozzle and its slot at the lower end of the cuvette flow cell with 70 % ethanol. Insert the close-loop nozzle.
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.7. Turn off the external temperature control system and the aerosol management system. 
SECTION: High-throughput isolation of nonreplicating persisters by FACS
46.8. Turn off the sorter. 
SECTION: Sample preparation for FACS
35.1. Remove the plunger of one syringe and fill the volume with ddH2O. Install the plunger and flush. (1/3)
SECTION: Sample preparation for FACS
35.2. Remove the plunger of one syringe and fill the volume with ddH2O. Install the plunger and flush. (2/3)
SECTION: Sample preparation for FACS
35.3. Remove the plunger of one syringe and fill the volume with ddH2O. Install the plunger and flush. (3/3)
SECTION: Sample preparation for FACS
35.4. Finally flush the homogenizer once with ice-cold HS buffer. Make sure there is no leakage or clogging.