CELL CULTURE AND MITOTIC ARREST
1| Mitotic arrest in adherent cell cultures.
TIMING 6 hours
i) Culture cells according to specific procedure for the cell type.
NOTE In our studies, CHO cells were grown in α-MEM containing 10% FCS. C166 and HT1080 cells were grown in DMEM containing 10% FCS. C166 and CHO cells are grown in 15 cm petri dishes and HT1080 cells are grown in T75 cell culture flasks.
ii) Replace media in approximately 75% confluent cultures. Allow cells to grow for 2 hours.
NOTE We recommend using a smaller volume of media than for culturing cells, i.e. 10ml in 15 cm dishes or T75 cell culture flasks.
iii) Add 0.1 µg ml-1 of Colcemid to cell cultures and incubate for 3 to 4 hours.
NOTE In adherent cultures, mitotic cells can be detected as cells that are less adherent and round up by microscopy. The time of incubation with Colcemid can be increased when working in cells that are cycling slowly.
CAUTION Colcemid is toxic. Wear gloves.
MITOTIC CELLS HARVEST AND CHROMOSOME ISOLATION
2| Mitotic shake off in adherent cell cultures.
TIMING 20 minutes
i) Collect mitotic cells by shake off.
NOTE The shake off procedure will depend on the cell type. CHO and C166 mitotic cells are grown in 15 cm petri dishes and harvested by rinsing the plates with their media for 3 to 4 times. HT1080 cells are grown in T75 flasks and harvested by tapping the flasks vigorously.
ii) Pool the cells together and aliquot in 50 ml tubes.
iii) Take a cell count.
3| Chromosome isolation.
There are several methods to isolate chromosomes in suspension. The method presented here is a version of the polyamine-based method for adherent cell cultures5. A more exhaustive examination of the various chromosome isolation procedures6 and procedures for preparation of chromosomes in suspension from suspension cells7-10 are available elsewhere. So far, we have only performed chromosome flow FISH using the polyamine method.
i) Spin down the harvested cells at 350 g for 5 minutes at room temperature.
ii) Aspirate the media from the tube.
ΔCRITICAL STEP It is important to remove as much of the cell culture media as possible, as it will interfere with the next step.
iii) Disturb the cell pellet by flicking the tube. Re-suspend the cell pellet by slowly adding hypotonic solution while swirling the tube. We added 5 ml of hypotonic solution when working with approximately 200,000 or less isolated mitotic cells and 10 ml with more harvested cells. Pool the cells together if they were in more than one 50 ml tube.
CAUTION The cells will swell and become more fragile in hypotonic solution, so we recommend manipulating gently.
iv) Incubate at room temperature for 5 to 15 minutes. We treated CHO cells in hypotonic solution for 15 minutes, C166 for 5 minutes and HT1080 for 12 minutes.
ΔCRITICAL STEP The time of hypotonic treatment required to swell the cells will depend on the cell type used.
v) During the incubation time, stain an aliquot of cells with a nucleic acid stain such as propidium iodide and calculate the mitotic index.
NOTE Mitotic cells can be differentiated from nuclei by their condensed chromosomes on the fluorescent microscope. The presence of nuclei will not interfere with the chromosome isolation procedure, but it is recommended to count the number of mitotic cells to calculate the number of chromosomes obtained in the isolation procedure.
vi) Spin down the cells at 350 g for 5 minutes at room temperature.
vii) Aspirate the hypotonic solution from the tube.
NOTE After successful hypotonic treatment, the cell pellet should be enlarged compared to step 3 ii. CAUTION Take care not to disturb the cell pellet as it tends to be loose after hypotonic treatment.
viii) Disturb the cell pellet by flicking the tube. Re-suspend the cell pellet by slowly adding freshly made, ice-cold chromosome isolation buffer. Approximately 1 ml of chromosome isolation buffer per 200,000 harvested mitotic cells was used.
ix) Incubate on ice for 15 to 30 minutes.
NOTE During this incubation time, the detergent contained in the chromosome isolation buffer will destabilize the cell membranes.
x) Vortex vigorously for 75 seconds to liberate chromosomes from mitotic cells.
ΔCRITICAL STEP The required time to vortex will vary depending on the vortex force and the cell type. When using a new cell type, we recommend to vortex for 45 to 60 seconds. Liberation of chromosomes in suspension can be controlled by taking an aliquot of the suspension and staining with propidium iodide. If a high percentage of mitotic cells are still intact, a longer time to vortex is required.
ΔCRITICAL STEP It is important to control for proper release of chromosomes in suspension. If too much vortexing time is used, some chromosomes in suspension will start to uncoil (can be observed as long strings and debris under the microscope). We found that chromosome preparations with uncoiled chromosomes (even if they are only a fraction) resulted in severe clumping and poor quality flow karyograms after the denaturation step of the FISH procedure.
PAUSE POINT Chromosomes preparations should be kept at least 3-4 hours (ideally overnight) at 4°C prior to proceeding to the next step. The chromosome preparations can be kept at 4°C for a few weeks.
HYBRIDIZATION OF CHROMOSOMES IN SUSPENSION
4| Set water bath to 80°C and pre-warm 100 µl of hybridization solution per condition tested.
ΔCRITICAL STEP From this point onwards, protect from light to prevent photobleaching of the fluorochromes.
5| RNAse pretreatment.
i) Treat chromosome suspensions with 100 U ml-1 of RNAse T1 for 20 minutes at room temperature (18-25°C).
NOTE Ideally, we use 1-10 million chromosomes per hybridization, although the experiment can be performed with lower numbers. This step is performed directly in the chromosome isolation buffer.
ii) Spin down chromosomes at 350 g for 5 minutes at 4°C
iii) Aspirate the supernatant.
ΔCRITICAL STEP Try to remove as much as the supernatant as possible as to not interfere with the next step. We recommend using a 200 µl tip.
i) Disturb the pellet by flicking the tube.
ii) Add 100 µl of hybridization mixture to the chromosome pellet. Mix very gently.
ΔCRITICAL STEP In our studies, we used a telomere probe (5'-Cy5-(CCCTAA)3-3') at a concentration of 0.3 µg ml–1, a mouse major satellite probe (5'-Cy5-GACGTGGAATATGGCAAG-3') at a concentration of 0.75 µg ml–1 and human L1.84 alpha satellite probe (5'-Cy5-GAGAATTGAACCACCG-3') at a concentration of 0.2 µg ml–1. Titration (figure 2) of each probe is important, as each PNA will have different properties.
iii) Denature the chromosome suspension by immersing in a water bath at 80°C for 5 minutes.
ΔCRITICAL STEP Denaturation time and temperature should be exact to prevent excessive damage and allow for optimal hybridization.
CAUTION Formamide is a poison and a mutagen. Wear gloves and work in a hazardous fume hood. Waste should be collected according to institutional rules and regulations.
iv) Hybridize at room temperature (18-25°C) for 60 minutes. Protect from light.
7| During the hybridization time, pre-warm at 37°C 1ml of wash solution per hybridization tube.
8| Excess probe washing.
i) Following hybridization, add 500 µl of wash solution to chromosomes in hybridization solution. Incubate for 5 minutes at 37°C.
ii) Spin down the chromosomes at 1675 g for 10 minutes at 4°C.
CAUTION Chromosomes should not pellet entirely when in 70% formamide (unless there are large clumps).
iii) Remove the supernatant, leaving behind 100 µl.
ΔCRITICAL STEP The centrifugal force required to pellet nuclei and chromosomes in 70% formamide is much higher than in isotonic aqueous solutions. The goal of the wash step following hybridization is to enrich chromosomes in the bottom fifth of the tube without creating a clear pellet since forcing chromosomes on top of each other will result in clumps that are difficult to resolve. Loss of material at this step is inevitable and it is important to find the right balance between acceptable looses and good single chromosomes suspensions for each cell type. We recommend using a p1000 tip to remove the largest part of the washing solution, and to finish with a p200 for more precision. It is important to leave behind the last 100 µl of solution.
iv) Repeat steps i to iii.
9| Re-suspend hybridized chromosomes in chromosome isolation buffer containing 40 ml-1 of chromomycin A3 and 2 µg ml-1 of Hoechst 33258.
PAUSE POINT The chromosomes should be stained for at least 4 hours prior to analysis. We usually keep the chromosomes at 4°C overnight.
FLOW CYTOMETRY ACQUISITION.
TIMING Variable, depending on sample concentration and number
10| Filter chromosome samples using 35 µm cell strainer cap tubes.
CAUTION Do not push chromosome suspension through the cell strainer, but rather tap the strainer tube gently to filter.
11| Sample acquisition on the Influx I.
i) Adjust the Cy5 PMT power to 50 mW.
NOTE We found that with our 125 mW 642 nM laser, a PMT power of 50 mW is optimal to detect weak Cy5 fluorescence signal above background while preserving the resolution of bright signal. When using a lower laser excitation power, PMT power can be further increased to detect populations with weaker fluorescence intensities from background, but it can be at the cost of resolution between populations of similar fluorescence intensities.
ii) Load the sample and adjust the PMT power (generally ==== 35-40 mW for Hoechst 33259 and ==== 45-50 mW for chromomycin A3 on our instrument) for collection of Hoechst 33258 and chromomycin A3 to obtain the best spreading of chromosome clusters for the sample analyzed.
iii) Analyze 50,000 to 100,000 (or more) events per sample. For all samples, we acquire data on pulse width, forward scatter, side scatter, Hoechst 33258, chromomycin A3 and Cy5.
PAUSE POINT Analysis can be performed anytime after flow cytometry analysis.
12| In our studies, we analyzed our data using FlowJo version 8.8.6.
i) Gate out debris on a pulse width versus chromomycin A3 plot (figure 3).
ii) Repeat on a pulse width versus Hoechst 33258 plot.
iii) Plot bivariate Hoechst 33258 versus chromomycin A3 flow karyograms and Cy5 versus chromomycin A3 density plots, as desired.