TITLE: A live-cell platform to isolate phenotypically defined subpopulations for spatial multi-omic profiling
AUTHORS: Tala O. Khatib, Angelica M. Amanso, Christina M. Knippler, Brian Pedro, Emily R. Summerbell, Najdat M. Zohbi, Jessica M. Konen, Janna K. Mouw, Adam I. Marcus

[DESCRIPTION]


Numerous techniques have been employed to deconstruct the heterogeneity observed in normal and diseased cellular populations, including single cell RNA sequencing, in situhybridization, and flow cytometry. While theseapproaches have revolutionized our understanding of heterogeneity, in isolation they cannot correlate phenotypic information within a physiologically relevant live-cell state with molecular profiles. This inability to integrate a live-cell phenotype - such as invasiveness, cell:cell interactions, and changes in spatial positioning - with multi-omic data creates a gap in understanding cellular heterogeneity. We sought to address this gap by employing lab technologies to design a detailed protocol, termed Spatiotemporal Genomic and Cellular Analysis (SaGA), for the precise imaging-based selection, isolation, and expansion of phenotypically distinct live cells. This protocol requires cells expressing a photoconvertible fluorescent protein and employs live cell confocal microscopy to photoconvert a user-defined single cell or set of cells displaying a phenotype of interest. The total populationis then extracted from its microenvironment, and the optically highlighted cells are isolated using fluorescence activated cell sorting. SaGA-isolated cells can then be subjected to multi-omics analysis or cellular propagation for in vitro or in vivo studies. This protocol can be applied to a variety of conditions, creating protocol flexibility for user-specific research interests. The SaGA technique can be accomplished in one workday by non specialists and results in a phenotypically defined cellular subpopulations for integration with multi-omics techniques. We envision this approach providing multi-dimensional datasets exploring the relationship between live cell phenotypes and multi-omic heterogeneity within normal and diseased cellular populations.




[GUIDELINES]
Reagent Set Up

Complete medium – Prepare RPMI 1640 (with and without phenol red) supplemented with 10% (vol/vol) FBS and 1% (vol/vol) Penicillin Streptomycin. Prepared media can be stored at 4 °C for two weeks. Maintaining sterile culture, remove necessary volume as needed and pre-warm aliquot to 37 °C before use (unless otherwise noted).
Flow buffer – Prepare fresh on the day of use by supplementing RPMI 1640 or appropriate base media (without phenol red) with 5% (vol/vol) dialyzed FBS. Alternatively, calcium free reagents can be used to reduce cell aggregation.

3D matrix embedment experiments
 

Recombinant basement membrane master mix – Thaw growth factor reduced recombinant basement membrane (often referred to as its tradename Matrigel, rBM) at 4 °C 30 min and maintain on ice to avoid polymerization at room temperature. Spheroids are embedded in 5 1798 rBM (in full media) and then plated onto a glass bottom dish. Note, the polymerization characteristics of rBM may change from lot to lot of Matrigel; as such, the concentration may need to be adjusted on a lot-specific basis. Additionally, cell secreted growth factors and cytokines will vary between lots of non-growth factor depleted Matrigel; growth factor reduced Matrigel leads to a more consistent composition (5).
Collagen I master mix – Keep stock collagen I (high concentration derived from rat tail) solution at 4 °C or on ice until ready for use. on ice, supplement stock collagen I with 10 % (vol/vol) PBS to a working concentration of 3 1798. Check pH of solution with pH strips and adjust pH to approximately pH 7.0. Keeping working solution on ice until ready to use. Do not store dilute solution long-term.
Collagenase/dispase cocktail: Suspend 100 mg of collagenase/dispase (C/D) in 1 mL of PBS or base media (without added growth factors or FBS) for a 100 1798 stock solution. Importantly, calcium is an important factor for enzymatic stability and activity in this cocktail; therefore, in the case of PBS, calcium supplemented PBS is required. Pipette gently to thoroughly resuspend. Sterilize with a 0.2 μm filter. Make aliquots of stock at 50-100 µL per aliquot. Freeze at -20 °C until ready for use.


Sample Preparation

Guidelines: This section describes our approach to handling a variety of cell lines and patient samples in culture. Non-adherent and 2D conditions are respective to those particular and potentially uniquely appropriate to the experimental questions and cell treatments. 3D tumor spheroid formation is described in detail by using the example human NSCLC H1299 cell line. See Table 2 for parameters to consider for optimizing the 3D invasion assay procedure to other cell lines and systems.

ADDITIONAL NOTES
Additional troubleshooting notes can be found in Table 5.

Table 5 I Troubleshooting table
 
      Step  Problem  Possible reason  Solution    18  Cells are unable to form spheroid  Low cell - cell adherence junction expression, low incubation time  Repeat centrifugation (step 19) and/or incubate for an additional 24 h.    33  Cells are shrinking; detaching from plate; swelling  Inadequate cell culture conditions on tabletop incubator  Ensure that the incubator is working at the appropriate temperature,   pressure, and CO2 level.    35  Poor imaging resolution  Scanning pixel size and/or line averaging amount is too low  Increase these imaging acquisition parameters to increase resolution.    37c  No fluorescent signal in the red channel  Laser intensity value is too low resulting in low to no photoconversion  Increase number of repetitions and/or bleach iterations. May need to   increase laser intensity.    37c  No fluorescent signal in the red channel  Laser intensity value is too high resulting in photobleaching ROI  Disregard ROI, decrease laser intensity, and select another ROI.    37c  Cell shrinking or swelling after photoconversion  Laser intensity too high resulting in phototoxicity  Disregard ROI, decrease laser intensity, and select another ROI.    37d  Low fluorescence in the red channel  Low photoconversion efficiency  Increase number of repetitions and/or bleach iterations. May need to   increase laser intensity.    44,45  Unable to degrade matrix  Enzyme concentration too low, inadequate incubation time  Increase enzyme concentration or incubation time. Agitate matrix with   pipette tip more frequently to encourage degradation.    47  Unable to degrade cell-cell junctions within spheroid  Enzyme volume or concentration too low, inadequate incubation time  Increase concentration, volume, or incubation time. Gently vortex to   encourage junction cleavage.    68  Number of cells recovered is higher than number of photoconverted cells  Off-target photoconversion due to inadequate ROI placement and/or   autofluorescence  Create a stricter ROI to ensure no off target or false positive   photoconversion of nearby cells. Some cell types emit autofluorescence,   ensure cytometer voltage settings are set to allow for enough separation   between those autofluorescent cells and those that were photoconverted.   Decrease laser intensity or time course on microscope to reduce off target   photoconversion.    68  Low cell viability post FACS  Inadequate sample preparation and/or maintenance  Keep cells on ice to slow intracellular metabolism and increase survival.   Avoid generating a dry pellet or air bubbles during processing. Air bubbles   may create a surface tension that is toxic to the cells. Avoid vigorous   vortexing and instead mix with gentle pipetting. If cell centrifugation is   necessary post FACS, apply low speeds (125 - 250 g RT).    72  Poor cell proliferation and propagation  Poor collection conditions; not enough cells; crucial growth factors not   present  Sort into culture media with at least 20% FBS to increase growth factors   and promote cell survival. Coat cultivation plates with protein to promote   cell adhesion. Plate cells on smaller surface area plate to facilitate cell -   cell communication to promote cell survival.  
 












[STEPS]
SECTION: Thawing and maintenance of cells
1. 
 
Prepare cell culture media as described in reagent set up.
SECTION: Thawing and maintenance of cells
2. Warm desired volume of culture media in 50 mL conical tubes at 37 °C in bead or water bath.

SECTION: Thawing and maintenance of cells
3. Prepare tissue culture appropriate laminar flow hood using UV light and wipe down all working surfaces with 70% ethanol (EtOH, vol/vol). Perform all cell culture within the laminar flow hood to maintain sterility. Use rigorous aseptic/sterile tissue culture technique where appropriate.
SECTION: Thawing and maintenance of cells
4. Rapidly thaw a vial of RPMI8226-Dendra2 or H1299-Dendra2 (either pal- or H2B) cells by gentle agitation in 37 °C water bath.

SECTION: Thawing and maintenance of cells
5. Decontaminate the vial by spraying with 70% (vol/vol) EtOH.
SECTION: Thawing and maintenance of cells
6. In the tissue culture hood, quickly open and transfer the vial contents to a 15 mL centrifuge tube containing 9.0 mL complete culture medium and spin at 125 x g, 5 min to pellet the cells out of solution. Speed and times may vary between cell lines.

SECTION: Thawing and maintenance of cells
7. Carefully remove the supernatant from above the pellet, taking care not to disturb the pellet. Resuspend the cell pellet with complete media and dispense into a 25 cm2 or a 75 cm2 (if working with cell concentrations greater than 1 x 106) culture flask. The density of the cells and volume of culture media may vary between cell lines.
SECTION: Thawing and maintenance of cells
8. Incubate at 37 °C and atmosphere of 95% Air, 5 % CO2. These conditions may vary by cell line.

SECTION: Thawing and maintenance of cells
9. Optional: when necessary, change media after 24 h to eliminate cells that do not survive thaw cycle.
SECTION: Thawing and maintenance of cells
10. Allow cells to recover from thaw (typically 2-3 days) and passage 1X prior to experiment set up.
SECTION: Thawing and maintenance of cells
11. Ensure media renewal occurs once every two days, or as appropriate for the cell line.
SECTION: Thawing and maintenance of cells
12. Passage cells at less than 70% confluency and ensure to collect all cells. 
 

SECTION: Thawing and maintenance of cells
12.1. For adherent cell passaging:
Collect conditioned/spent media in 50 mL conical tube.
Wash 1X with PBS and add to the conditioned media.
Detach cells with method of choice (trypsin, EDTA, Accutase). Different cell types may require distinct cell detachment mechanisms; therefore, it is best to determine the best passaging conditions for your system.
Neutralize trypsin enzyme with an appropriate trypsin neutralizing solution (TNS) at a 1:1 trypsin to TNS ratio. While some TNS product utilize serum (FBS or otherwise) to provide excess substrate for the trypsin to enzymatically digest protein molecules, an alternative – particularly for cells cultured serum free – is the use of a TNS product consisting of a 1X PBS solution containing 0.0125% (vol/vol) soybean trypsin inhibitor.
Pipet additional complete media onto plate to wash and collect all cells from plate and add to the 50 mL conical tube.
Centrifuge conical tube at 125-250 x g, 5 min, centrifuge speed depends on the cell line.
Resuspend in 3 mL of complete media.
Count cells using 0.4% Trypan blue solution at a 1:1 ratio with hemocytometer.


SECTION: Thawing and maintenance of cells
12.2. Non-adherent cells do not require enzymatic cleavage from cell plate and can simply be collected from plate at time of experimentation. Cell concentrations may vary by plate or cell size.
SECTION: Option A: Non-adherent sample preparation for photoconversion
13. 
 
Collect and count RPMI8226-Dendra2 cells using 0.4% Trypan blue at a 1:1 ratio with hemocytometer.
SECTION: Option A: Non-adherent sample preparation for photoconversion
14. 0.5 × 106 cells are isolated from the bulk sample and spun down at 125-250 x g, 5 min, centrifuge speeds may vary by cell line. Number of cells will vary by plate and cell size.

SECTION: Option A: Non-adherent sample preparation for photoconversion
15. Resuspend cells in 200 µL phenol-red free complete media and transfer to 8-well glass bottom slide chamber. Cell number is dependent on cell morphology and well dimensions.
 

SECTION: Option B: 3D cell culture sample preparation for photoconversion
16. 
 
To generate spheroids, plate 3,000 H1299 cells in 200 µL (1.5 × 104 cells per mL) in a low adherence 96-well plate. Create 18 spheroids per condition to embed 10 – 12 spheroids in matrix. Excess spheroids are created to account for shape and stability deformities during incubation period for spheroid formation (Table 2). The cell number may be adjusted for different cell lines and spheroids of different diameters (Table 2).

Table 2 I Parameters for spheroid formation 
      Parameters  Optimization  Examples  Steps    Seeding density  Spheroid density depends upon cell size, shape, morphology, and rate of   proliferation. Various densities should be screened to achieve ~ 500 μm in   spheroid diameter upon embedding into matrix. Importantly, the larger the   cell number, the larger the spheroid, the greater oxygen differential between   the external cells and the cells internal to the 3D structures.  3000 cells/well (H1299, 4T1) 1000 cells/well (A375)  Steps 16 – 18    Nanoparticle contamination  Sterile 96-well plates and/or   sterile pipet tips are often contaminated with sterilized nanoparticles that   can become embedded within a spheroid and deform its shape. 1.5X spheroids   are created to account for unusable spheroids.  Steps 16 – 25    Cell adherence  Heterogeneous cells can express distinct adherence junction profiles to   regulate cell – cell junctions and cell – matrix adhesion/interactions.   Centrifugating 96-well plate places cells in the center of the well near one   another to promote cell – cell junction formation. Upon spheroid formation,   different matrices can be screened to determine the ability for cell – matrix   adhesion formation and interactions.  rBM (H1299, A375) Collagen I (4T1)  Step 18    Time  After centrifugation, spheroid formation requires a 24 h or more   incubation time. Cells can be screened to determine optimal incubation time   to maintain both spheroid integrity during the embedding process and cell   viability after.  72 h (H1299, 4T1, A375)  Step 18  
 
SECTION: Option B: 3D cell culture sample preparation for photoconversion
17. Centrifuge plate at 450 x g, 10 min to collect cells at the center of each low absorbance round-bottom well.

SECTION: Option B: 3D cell culture sample preparation for photoconversion
18. Incubate cells for 4320 min for cell – cell junction formation within the spheroid. 
 

SECTION: Option B: 3D cell culture sample preparation for photoconversion
19. Prior to the day of embedment, snip an experimentally appropriate number of 1000 μL and 200 μL pipet tips for embedding spheroids into matrix. Autoclave to re-sterilize tips and allow to cool to room temperature.
SECTION: Option B: 3D cell culture sample preparation for photoconversion
20. A standard light microscope can be used prior to spheroid removal from the round bottom plate to validate spheroid integrity has been maintained (no debris contaminating or altering spheroid) (Table 2). Depending on the cell line, spheroids may not be spherical in shape. While sphericity is not necessary for transfer, spheroid stability is necessary for extraction from the round bottom plates and successful embedment. Non-stable spheroids/structures may fall apart during the process.
SECTION: Option B: 3D cell culture sample preparation for photoconversion
21. Collect usable spheroids with tip-snipped 1,000 μL pipet tips into 1.7 mL microcentrifuge tube - one experimental group per tube. The same snipped-tip can be used for all spheroids within the same experimental group. Pipet 8 spheroids per 1.7 mL microcentrifuge tube. The number of spheroids included can be increased or decreased depending on the experiment.
SECTION: Option B: 3D cell culture sample preparation for photoconversion
22. Allow the spheroids to sink to the bottom of the microcentrifuge tube. Remove excess media taking care not to pipet up the spheroids. Remove single and detached cells by washing spheroids with 1 mL of complete medium 2X by pipetting media along the edges of the tube in a circular motion, allowing the spheroids to sink, and removing excess media. 
 

SECTION: Option B: 3D cell culture sample preparation for photoconversion
23. Collect spheroids in 100 µL 3D master mix using tip-snipped 200 μL pipetmen and plate in 35-mm glass bottom dish.
SECTION: Option B: 3D cell culture sample preparation for photoconversion
24. Use a second unsnipped 200 μL pipette tip to carefully spread master mix to cover entire glass surface.
SECTION: Option B: 3D cell culture sample preparation for photoconversion
25. With a third unsnipped 200 μL pipette tip, carefully move and spread spheroids for equal distribution. 
 

SECTION: Option B: 3D cell culture sample preparation for photoconversion
26. Allow >30 min for matrix to polymerize. After complete polymerization, add 1.5 mL pre-warmed complete medium and incubate. 
 

SECTION: Option B: 3D cell culture sample preparation for photoconversion
27. Image every 24 h if monitoring surface area or circularity (Table 3).

Table 3 I 3D spheroid invasive area and circularity quantification
 

SECTION: Option C: 2D cell culture sample preparation for photoconversion
28. 
 
Passage cells at < 70% confluency and plate cells on glass bottom plate for photoconversion the following day. Cell number will be dependent on plate size and cell morphology. Cell confluency will be dependent on experimental question.
SECTION: Option C: 2D cell culture sample preparation for photoconversion
29. If distinct phenotypes are present temporally upon treatment with drug or other additive factor, adjust time course and confluency to match these criteria.
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
30. 
 
Prepare stage top incubator to maintain standard tissue culture conditions, typically 5% CO2 at 37 °C. These conditions may vary by cell line and are utilized to facilitate typical cellular phenotypes under defined experimental conditions. 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
31. Fill stage top incubator with enough autoclaved distilled water to maintain humidity and compensate for evaporation during imaging. Allow the incubator to equilibrate for at least 120 min.

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
32. Turn on the computer, microscope, scanner, and laser power source. Photoconversion requires three laser lines: 405 nm for photoconversion, 488 nm for visualization of Dendra2-green (excitation and emission peaks: 490/507 nm), and 561 nm for visualization of Dendra2-red (excitation and emission peaks: 557/573 nm). Alternatively, can use 543- or 568 nm laser lines in place of 561 nm for visualization of Dendra2-red (emission spectral ranges: 570-670 nm).
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
33. Place the plate with cells inside stage top incubator and incubate for 30 min to allow cells to equilibrate to microscope conditions.
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
34. Open laser configuration window and turn on the Diode 405 nm (UV) laser, Argon laser (visible), and the DPSS 561 nm laser lines. 
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35. Image acquisition set-up.
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.1. Turn off resonant scanning. 
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.2. Set the scanning parameters to the XYT (XY Time) mode. Time is necessary for photoconversion (Step 37d).
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.3. Set scanning pixel size to be 1024 x 1024 or lower. 
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.4. Use the default scanning speed (400 – 800 Hz). This is the acquisition rate of pixels per second.
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.5. Determine the zoom factor empirically. 
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
35.6. Set line averaging at 2 and change as needed depending upon resolution needs or scanning speed requirements.
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36. Configure laser parameters.
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36.1. Open the laser configuration window for the 405 nm, 488 nm, and 561 nm laser lines.
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36.2. Set the 488 nm and 561 nm laser lines to sequential scanning to avoid emission spectra crosstalk. Use the ‘between line’ scanning feature so live cell movement does not lead to signal artifact between channels. 
 


SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36.3. Visualize the sample and adjust the objective to the desired focal plane.
SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36.4. Use live view to adjust the detector, gain, and laser intensities for optimal exposure.
 

SECTION: Confocal imaging and photoconversion: Microscope and laser set-up
36.5. Use the photomultiplier tube (PMT) detector for photoconversion because during the bleach sequence, high intensity illumination targets the region of interest (ROI). The hybrid detector (HyDs) may switch off to protect the ROI from photon overload, resulting in loss of the post-bleach sequence.
SECTION: Confocal imaging and photoconversion: Photoconversion
37. 
 
The photoconversion parameters set-up utilizing the FRAP interface.

 

SECTION: Confocal imaging and photoconversion: Photoconversion
37.1. Turn the zoom-in feature on to allow for precise illumination and ROI selection. 
SECTION: Confocal imaging and photoconversion: Photoconversion
37.2. Set the background to zero to ensure that the area outside of the ROI is not exposed to background light. This will decrease the potential for false positive cell photoconversion near the ROI.
SECTION: Confocal imaging and photoconversion: Photoconversion
37.3. Set the 405 nm laser intensity for photoconversion. This is dependent on cell type and experimental conditions. It is important to test various intensities to determine which intensity maintains cell viability while ensuring complete photoconversion for cells within the ROI (Table 4, Fig. 5B – D). 
 

Table 4 I Photoconversion time course guidelines 
      These criteria were established after extensive screening of   each cell line and culturing condition. Similar screening should be done   prior to establishing a photoconversion regimen for other experimental   conditions.    Procedure –   Timing 1 - 4 h      1. Open the   405 nm shutter and adjust laser power to respective intensity dependent on   experimental conditions (see below). Laser intensity may vary by experiment   or microscope.    2. Turn down   all other laser lines to zero as they will not be in use during   photoconversion.    3. Set the   number of prebleach, bleach and postbleach intervals in the time course   frame. Of note, these settings are dependent on experimental conditions and   can be enhanced for optimization.    4. Set ROI   and run experiment. Continue as needed until all ROI are photoconverted.    Experimental conditions  Non-adherent  3D spheroid  2D monolayer    405 nm laser intensity for   photoconversion  5%  15%  10%    Repetitions  1  1  1    Prebleach interval  1  1  1    Bleach 3 - 5 sec interaction  1  2  3    Postbleach intervals  1  1  1  
 
SECTION: Confocal imaging and photoconversion: Photoconversion
37.4. Establish a time course for the number of prebleach, bleach, and post-bleach frames. These values are dependent on the experimental design. Optimize the time course by testing various conditions for each sample in study (Table 4). During these screening stages, monitor cells for any unhealthy signs, such as swelling or shrinkage.
 

SECTION: Confocal imaging and photoconversion: Photoconversion
38. Select the phenotype-driven ROI and begin the established time course for photoconversion.
SECTION: Confocal imaging and photoconversion: Photoconversion
39. Repeat steps 37 – 38 as necessary until all user-defined cells are photoconverted.
SECTION: Confocal imaging and photoconversion: Photoconversion
40. Assess efficiency of photoconversion within the evaluation menu on the user interface (Fig. 4B, C). The integrated density relative fluorescence values pre- and post-photoconversion are shown. The Dendra2-green should significantly decrease and Dendra2-red should significantly increase after photoconversion (Fig. 4B – E).
 

SECTION: Fluorescence-activated cell sorting (FACS) - Option A: Non-adherent sample preparation for FACS
41. 
 
Centrifuge non-adherent cells at 125-350 x g, 5 min. Centrifuge speeds may vary by cell line.

SECTION: Fluorescence-activated cell sorting (FACS) - Option A: Non-adherent sample preparation for FACS
42. Proceed to step 51.
SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
43. 
 
Dilute stock Collagenase/Dispase (C/D) cocktail in sterile media without serum for a working concentration between 1 – 5 mg/mL. For digestion of rBM 1 mg/mL, is sufficient. For digestion of type I collagen, a higher concentration is recommended. Enzyme concentration can be increased if the matrix is difficult to degrade.
SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
44. Option 1: Mince matrix into quarters and place into a microcentrifuge tube with 3 – 4X volume of the minced matrix with the working stock of C/D. Place in a 37 °C incubator. Lightly vortex every 5 – 10 min until matrix is digested.
SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
45. Option 2: Digest the matrix directly in the glass bottom dish. Remove all media prior to adding 3 – 4X volume of the working stock of the C/D digestion buffer. Place dish in a 37 °C incubator. Pipette gently every 5 – 10 min until matrix is digested and cells are released. 
 

SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
46. Centrifuge cells at 150-300 x g 5 – 10 min.

SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
47. Resuspend in trypsin (or similar proteolytic enzyme suitable for cleaving cell – cell junctions) to further digest spheroids and cell clusters into single cells. A standard light microscope can be used to visualize the formation of single cells. 
 

SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
48. Inactivate trypsin with TNS at a 1:1 ratio and centrifuge cells at 125-250 x g, 5 min. Centrifuge speed may vary depending on cell line.

SECTION: Fluorescence-activated cell sorting (FACS) - Option B: 3D cell culture sample preparation for FACS
49. Proceed to step 51.
SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
50. 
 
Repeat steps 12.1.1 – 12.1.6 for cell collection.
SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
51. Wash cells 1X by resuspending the cell pellet in 5 mL of flow buffer.

SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
52. Centrifuge sample for 250 x g, 5 min to pellet cells.

SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
53. Gently resuspend approximately 1.0 × 106 cells or less into 100 µL flow buffer. For samples with more than 1.0 × 106 cells, resuspend in a larger volume to avoid cell aggregation or flow cytometer clogging.
SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
54. Add 5 µL per 100 μL cell suspension of Annexin V conjugate (per manufacturer recommendations) and incubate for 10 min at Room temperature. Protect samples from light from this point forward.
 


SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
55. Add 200 µL flow buffer per 100 μL solution. Gently pipette to mix and transfer to a 5 mL flow tube with a cell strainer. Place sample on ice for transport to the FACS sorter.

SECTION: Fluorescence-activated cell sorting (FACS) - Option C: 2D cell culture sample preparation for FACS
56. Prepare a tube with 1 mL of complete culture medium for collection of cells after FACS. Prior to sorting, run each sample through cell strainer flow tube cap to decrease cell aggregation.

SECTION: FACS set up
57. 
 
Ensure waste tank is empty and sheath tank is at least 50% full. Sheath tank should be tightly closed to allow for pressure build up in the system.
SECTION: FACS set up
58. Power on the computer and activate the compressed airline to at least  .

SECTION: FACS set up
59. Switch on the cell sorter and start the sorting software, ensuring the computer has successfully connected to the FACS sorter.
SECTION: FACS set up
60. Initiate the fluidics start-up and follow the popup tutorial to complete required steps. This start-up will take approximately 10 min.

SECTION: FACS set up
61. Remove the closing nozzle from the flow cell assembly and attach the appropriate nozzle for your experimentation.
 

SECTION: FACS set up
62. Activate the stream and stabilize to desired flow rate. 
SECTION: FACS set up
63. Calibrate the cytometer with cytometer set-up software and tracking beads. Set drop delay using Accudrop beads. This calibration will take approximately 20 min.

SECTION: FACS set up
64. Sterilize the cytometer interior with 70% (vol/vol) EtOH.
SECTION: FACS set up
65. Sterilize the sample line with 10% (vol/vol) bleach and rinse with water.
SECTION: FACS collection
66. 
 
Use lasers blue (B) 488 nm, yellow/green (YG) 561 nm, red (R) 633 nm, to excite and visualize the following, respectively: Dendra2-green – bandpass filter: B 530/30, Dendra2-red – bandpass filter: YG 582/15, Annexin V (Alexa Fluor 680) – bandpass filter: R 710/50. Additionally, Annexin V pacific blue (laser line: violet 405 nm, bandpass filter: 450/50) can be used in conjugation with Dendra2. Due its low intensity, the 405 nm and 488 nm lasers will not photoconvert Dendra2 while in the FACS sorter.
SECTION: FACS collection
67. Prepare a workbook to collect live, single cells.
SECTION: FACS collection
68. Perform the respective controls (as described in experimental design, Fig. 5A) and then flow sort cells positive for Dendra2-red into the collection tube prepared in Step 56 (Fig. 5A). These cells are the phenotypically driven, user-defined cell subpopulation of interest. 
 

SECTION: FACS collection
69. Process, culture, or store the collected cells based on the analysis of interest (Table 1, Fig. 2, Fig. 6).

Table 1 I Example downstream applications of SaGA-isolated subpopulations 
      Experimental approach  Application  Potential outcome    Immediate isolation  In vitro  Cell lysis for immediate contents extraction (i.e., protein, RNA, DNA,   and ribosomes)  Targeted transient expression profiling via immunoblotting, qPCR, etc.          Unbiased transient expression profiling via ATACseq, RNAseq, Riboseq,   etc.    Long-term cultivation in vitro  In vitro  Cell behavior, signaling, etc.  Stable phenotype identification, stable subpopulation generation,   determination of cooperative phenotype between subpopulations, targeted   expression profiling and unbiased multi-omic analysis.      In vitro  Introduction to model organism  Stable phenotype identification, determination of cooperative phenotype   between subpopulations, targeted expression profiling and unbiased multi-omic   analysis.  
 
SECTION: Downstream analysis post-FACS
70. 
 
To sort cells for immediate bulk -omic analysis, collect the same number of cells for each photoconverted population into the desired lysis buffer (such as DNA or RNA lysis buffer) (Table 1).
SECTION: Downstream analysis post-FACS
71. To sort for immediate single cell -omic analysis, sort single photoconverted cells into individual wells of a 96-well plate with desired lysis buffer and immediately place in a negative 80°C freezer (Table 1).
SECTION: Downstream analysis post-FACS
72. For cell propagation and long-term phenotypic analysis, sort cells into complete growth medium (Table 1). Culture and passage the cells as routinely done for further experimentation.