TITLE: Metaphase_spread_and_DNA_FISH_cell_lines
AUTHORS: jingting

[DESCRIPTION]
Cytogenetically detect single-cell ecDNAs in cell lines via staining/homologous DNA hybridization and fluorescence microscopy -- fluorescence in situ hybridization (FISH).

[STEPS]
SECTION: Introduction
1. Cytogenetically profile single-cell DNAs through metaphase karyotyping by DNA FISH.
SECTION: Material
2. Methanol/glacial acetic acid 3:1 -- Prepare fresh in a fume hood prior to use
Hypotonic stock solution: 0.075M KCl (gibco, ref. 10575-0821)
2 X SSC (Saline-Sodium Citrate) stock solution (/0.05% TWEEN20)
0.4 X SSC stock solution
Total probes : hybridization buffer = 1 : 4 (e.g. for a master mix of 10 uL from 2 different probes, add 1 uL probe 1 + 1 uL probe 2 to 8 uL hyb buffer) -- prepare fresh

SECTION: Procedure
3. Prepare metaphase spreads
SECTION: Procedure
3.1. Grow cells to 60-80% confluency.
SECTION: Procedure
3.2. Arresting cells at metaphase: Replace old media with media with 0.1 µg/mL Colcemid diluted from 10 µg/mL stock solution (Karyomax from Gibco. Ref#15212-012), (e.g. 100 µL of 10 µg/mL Colcemid/10 ml medium) and incubate O/N (depending on the cell doubling time).
SECTION: Procedure
3.3. Warm up 0.075M KCl in 37℃ water/bead bath.
SECTION: Procedure
3.4. Examine if cells are mostly mitotic that round up under the inverted microscope.
SECTION: Procedure
3.5. Transfer the colcemid-spiked media to a 15 ml conical tube. Wash cells with 5 ml PBS. Transfer the PBS wash to conical.
SECTION: Procedure
3.6. Add 1-2 ml trypsin (depends on cell density) to the cells in a 10 cm dish and digest for 3 min at 37℃.
SECTION: Procedure
3.7. Examine cells under the inverted microscope (optional). Quench the digestion by adding 3-5 ml colcemid-spiked media when most cells start to lift off (media should be at least 3 times the volume of trypsin). Gently pipette up and down 10x to flush off adhered cells.
SECTION: Procedure
3.8. Transfer the collected cells to the remaining colcemid-spiked media and centrifuge at 400x G for 4 min. Aspirate the supe. 
SECTION: Procedure
3.9. Wash cells with PBS: If cells <6 million, add 1 ml PBS; if cells ≥ 6 million, add n mL PBS, n = #cell/6 million rounded up (e.g. 2 tubes for 8 million). Gently pipette 10x to resuspend. Transfer the suspension to one or n 1.5 ml microcentrifuge tube(s). Centrifuge at 5000 rpm for 2 min and aspirate the supe.
SECTION: Procedure
3.10. Swelling cells: Add 600 µl pre-warmed 0.075M KCl dropwise down the side and resuspend by tapping/very gentle pipetting --> incubate for 15 min in 37℃ water/bead bath (15 min is the optimal time for swelling. Swollen cells are brittle – no vortex or pipetting hereafter. Cells must be sufficiently swollen to burst on microscope slides, whereas with excessive swelling, cells break in fixative and release cytoplasmic contaminants). Prepare fresh fixative in a fume food while waiting: ≥ 2.4 ml/pellet. Optional: place fixative on ice.
SECTION: Procedure
3.11. Add 600 µl Carnoy’s fixative (use 100-300 and 800-1000 µl for hundreds of K cells and ≥ 7 million cells, respectively) dropwise down the side to quench the reaction and immediately centrifuge at 5000 rpm for 2 min and aspirate/pipette the supe till a few drops (100-200 μL) left.
SECTION: Procedure
3.12. Fixing cells: Resuspend pellets in remaining solution by gently tapping/flicking the bottom of the tube till no clumps. Add (pre-chilled) 600 µl Carnoy’s fixative dropwise down the side and flick to mix well. Centrifuge at 5000 rpm for 2 min. Aspirate/Pipette until a few drops left.
SECTION: Procedure
3.13. Repeat step 11 twice.
SECTION: Procedure
3.14. Centrifuge and resuspend pellets in the remaining fixative. Add 300-1000 µl fixative depending on pellet size for a slightly milky suspension (over-concentrated cells are hard to profile individually and impair image quality). Cells can be stored in fixative at -20℃ for an extended time.
SECTION: Procedure
4. Prepare microscope slides
SECTION: Procedure
4.1. Prepare slides on a clean, dust-free surface (e.g. a clean slide box). Keep the sample side clean -- NO TOUCHING/SCRATCHING. Dropped metaphase spreads must be processed within 2 days to prevent chromosome degradation. 
SECTION: Procedure
4.2. Addditional washing steps for pre-made metaphase samples:
Thaw -20℃ metaphase samples at RT.
Prepare fresh Carnoy’s fixative in a fume hood while waiting.
Repeat step 10 twice or more till the supernatant is clear. 
SECTION: Procedure
4.3. Prepare 200-250 ml H20 in a 400 ml beaker. Microwave for 1-1.5 min for heated vapor.
SECTION: Procedure
4.4. Mix the suspension well by flicking/tapping. Pipette up 10 µL.
SECTION: Procedure
4.5. Place the slide over the hot water for humidity until tiny droplets are formed. Immediately drop the 10 µl suspension from height (an arm's stretch) to the moist slide. Re-humidify if the slide dries before drops hit.
SECTION: Procedure
4.6. Briefly hover the slide above the beaker for 2-3 s to further spread chrs by humidity.
SECTION: Procedure
4.7. Age/Dry at room temp (20-25℃) 1h - overnight in a closed environment (e.g. slide box/drawer...).
SECTION: Procedure
5. DNA FISH
SECTION: Procedure
5.1. Minimize exposure of fluo probes to light to limit photobleaching. Photobleaching impairs fluorescence and image contrast.
SECTION: Procedure
5.2. Preparations: Prepare 4 Coplin jars for 2X SSC, 70%, 85%, and 100% ethanol, enough to submerge metaphase spreads. Pre-warm DNA FISH probes in 37℃ water/bead bath; Set heat block to 70℃ based on the physical thermometer. Fill grooves in humidified chamber with dH2O and set to 37℃ --> close the lid.
SECTION: Procedure
5.3. Dehydration: briefly equilibrate spreads in 2X SSC (~3 sec) and immediately transfer to ascending alcohol gradients, 2 mins w/ lid on: 70% --> 85% --> 100% ethanol --> air-dry. While air-dry, prepare 10n µL of 1:5 probe master mix in hybridization buffer, e.g. In a colored PCR tube, add 2 µl of pre-warmed probes (Empire Genomics, stored in -20℃ protected from light), including all probes types, to 8 µl hybridization buffer (e.g. 1 µl each for 2 types of probes) and mix well by 3 pulses of vortex followed by a brief spin. For just DAPI staining, start from 28.
SECTION: Procedure
5.4. Add 5 µL of probe mix/slide onto the middle of the chromosome spread (NO TOUCHING/SCRATCHING!). Cover the spread with a 22x22 mm coverslip, taking care not to have bubbles or press too hard or slip. Push bubbles out using a pipette tip.
SECTION: Procedure
5.5. Co-denaturation: Immediately incubate the slide in the dark on a 70-72℃ heat block for 2 min (Heat exceeding 3 mins and/or 75℃ will damage chromosome morphology).
SECTION: Procedure
5.6. Probe hybridization: Incubate the slides at 37℃ O/N (16-20 h) in the pre-set dark humidified chamber. Humidity keeps the probe mix from drying out. Elongated hybridization leads to non-specific binding of FISH probes.
SECTION: Procedure
5.7. After hybridization: Prepare 3 Coplin jars containing fresh 0.4X SSC, 2X SSC + 0.05% tween20, and 2X SSC.
SECTION: Procedure
5.8. Post-hybridization wash: Gently lift the coverslip tweezing a corner -- no scraping! Wash slides in 0.4X SSC and 2X SSC/0.05% Tween20 for 2 min each with shaking for 10-15 s. Wash briefly in 2X SSC.
SECTION: Procedure
5.9. DAPI counterstaining: Remove excess buffer by wicking slides and wipe back of the slide clean using a paper towel. Add 1 drop of SlowFade Diamond Antifade Mountant with DAPI and seamlessly seal a 24x60 mm coverslip with nail polish.
SECTION: Procedure
5.10. Alternative DAPI staining: add 10 µl of 1:1000 or 200 µl of 1:10000 DAPI solution per slide and stain for 2 min in the dark. Agitate briefly in 2X SSC to remove excess DAPI. Remove the buffer as much as possible and wipe the back with a paper towel (watery liquid mixed with mounting media changes optimal refractive index for imaging).
SECTION: Procedure
5.11. Add 30 µl of VectaShield mounting medium (non-hardening with anti-fade) (Optional for just DAPI staining). Seal a 24 X 60 mm coverslip with nail polish.