Protocol to analyze chromatin-bound proteins through the cell cycle using Chromoflow flow cytometry

Summary Chromatin-bound proteins have been conventionally measured through subcellular fractionation followed by immunoblotting or by immunofluorescence microscopy. Here, we present Chromoflow, a protocol for the quantitative analyses of protein levels on chromatin in single cells and throughout the cell cycle using flow cytometry. We describe steps for harvesting cells and for nuclear extraction, and a barcoding strategy to multiplex samples from different conditions that reduces antibody staining variability and eliminates the need for normalization.1,2 We then detail procedures for data acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Alonso-Gil et al. (2023).3

1. Barcoding staining.Increasing concentrations of Pacific Blue dye will allow sample multiplexing, which reduces staining variability and facilitates comparisons among conditions.Four different conditions will be barcoded for this particular example: conditions 1, 2, 3 and 4. See Table 1. 2. Antibody staining.Simultaneous staining with primary antibodies from different host species (e.g., rabbit (rb), mouse (ms), goat) can be performed in the barcoded samples.Secondary antibodies must be from a different host species (e.g., donkey [dk]) and conjugated to fluorophores that are excited by different lasers (in this panel, Alexa Fluor 488 (AF488) and Cy3).3. Compensation controls are important to correct for spillovers from one fluorophore into secondary channels due to spectral overlap.Thus, single color staining controls must be included in the design.4. DNA content staining.In this particular panel design, we have chosen ToPro3.
Note: Laser and filters correspond to a BD LSRFortessa cell analyzer.They can be different depending on your panel design.Other fluorochromes can be read with different laser/filters.Note: Compensation is a basic process in analyses of flow cytometry data.The compensation strategy will depend on the lasers and filters of the cytometer used to detect the fluorophores used in your experiment.Several tutorials about compensation can be found in the internet or as part of the software used for analyses.We recommend that you discuss the compensation strategy of your experiment with an experienced user or person in charge of the Flow Cytometry Facility.

Note:
We have omitted a compensation control for Pacific Blue, as the spillover is minor (12% on B_525/20).See Figure S1.Working solutions to be prepared fresh for each experiment Timing: 30 min 7. Prepare extraction buffer as described under materials and equipment and keep it on ice.
CRITICAL: Always prepare a fresh dilution of Igepal CA-630.
8. Prepare staining buffer as described under materials and equipment and keep it at 25 C (room temperature, RT).
Note: Stock solutions of Sodium or Potassium phosphate may precipitate in the cold.Remember to heat and stir until complete dissolution before using it to prepare the staining buffer.9. Prepare a Pacific Blue working solution and dilutions as described under materials and equipment when nuclei are extracted (see step 13).Keep them at 25 C (RT).
CRITICAL: Make very precise dilutions.It is advisable to use pipets that have been recently calibrated.Note: Keep on ice and discard after use.
Note: Remember that you will need 1 mL per 2 3 10 6 cells.Scale as required depending on the number of samples.
Note: Stock phosphate buffer 0.1 M can be prepared either from 1 M Sodium or Potassium phosphate solutions.
CRITICAL: Igepal CA-630 concentration in extraction buffer will depend on the cell type.Some cells (e.g., HeLa, MCF10A or HCT116) require 0.1% detergent for successfully extract nuclei while others, such as mouse embryonic stem cells (mESC), are extracted with 0.07%.
Note: Keep at 25 C (RT) and discard after use.
Note: Scale as required depending on the number of samples.

Pacific Blue
Prepare a working solution (0.018 mg/mL) and keep it at 25 C (RT): 2 mL from a stock solution (5 mg/mL, stored at -80 C) + 553.5 mL DMSO Make very precise dilutions in eppendorf tubes and keep them at 25 C (RT):  Note: In this particular example, 2 double stainings of 4 conditions multiplexed in a barcoded sample will require 6 single control colors.Total number of tubes: 14 (2 stainings x 4 conditions = 8, plus 6 single stainings as control samples.See Figure 1.
Note: Cell confluency is very important.Asynchronous cell cycle analyses require no more than 70% confluency.Fully confluent plates will probably enrich the cell cycle profile in G1 phase.

Timing: 1.5 h
This step explains how to treat cells in order to remove the soluble components and leave chromatinassociated proteins.
5. Add 1 mL of extraction buffer to each 2 3 10 6 cell pellet.Pipet up and down to resuspend cells without making bubbles.See Figure 2. 6. Place samples back on ice for 5 min.7. Add 27 mL of 37% formaldehyde solution to the cell suspension (1% final concentration) and incubate for 1 h on ice.8. Fill the tube with PBS-1% FBS to stop the reaction.9. Centrifuge (5 min at 0.2 RCF, 4 C) and pour off the supernatant.10.Resuspend the extracted nuclei in the remaining PBS-1% FBS.See troubleshooting 1. 11. Transfer nuclei into a 1.5 mL eppendorf tube.12. Centrifuge (5 min at 0.8 RCF, 25 C) and strictly remove all supernatant using a micropipettor.
CRITICAL: Extraction has to be strictly timed (5 min incubation with extraction buffer).If you have more than 4 tubes (equal to one final barcoded sample), perform several rounds of extraction with no more than 4 tubes per round.
CRITICAL: One must be very careful in order not to lose nuclei when transferring them from falcon to eppendorf tubes (steps 10-12).CRITICAL: The proportion of 1 mL of extraction buffer for 2 3 106 cells is very important.Otherwise, extraction may fail.
Note: Extraction buffer is prepared fresh for each experiment and it must be stored on ice.
Optional: Steps 5-7 are required to analyze chromatin-associated proteins.To analyze total protein content, skip steps 5-7 and instead fix each 2 3 10 6 cell pellet with 1 mL cold EtOH-70% dropwise.Incubate for 2 h at -20 C and continue to step 8.In case of proteins tightly bound to chromatin, a more stringent extraction can be performed.After step 6 and before fixation, add 20 mL of 5 M NaCl to each tube.This will increase NaCl concentration in 100 mM.Incubate for 5 more minutes on ice and proceed to step 7.  Note: Staining buffer is prepared fresh for each experiment and it is stored at 25 C (RT).

Barcode the samples
Note: See primary and secondary antibody concentrations for this particular example in key resources table.
Note: Primary and secondary antibodies should have been titrated beforehand to find adequate staining conditions.Recommended dilution for immunofluorescence staining is a good starting point.
Note: Additional washing steps can be performed after primary and secondary antibody staining by repeating steps 22-23 and 25-26, respectively.

Timing: Depending on the number of samples (approximately 1.5 h)
In this step, data are acquired at the flow cytometer and saved for subsequent analyses.
29. Acquire data on BD LSRFortessa or your flow cytometer of choice.30.Gate a uniform population from Forward Scatter Area (FSC-A) and Side Scatter Area (SSC-A).31.Gate single cell cycles from DNA content-A (ToPro3-A) vs.DNA content-H (ToPro3-H).32.Represent single cells in SSC-A vs. Pacific Blue-A to gate the 4 conditions in barcoded samples.33.Represent DNA content-A (ToPro3-A) vs. your antibody of interest-H (Cy3/AF488-H) and adjust voltages on a linear scale.34.Record at least 10,000 events for each condition in barcoded samples.35.Acquire and record 20,000-30,000 events for control samples in order to compensate fluorescence spillover.See step 44 in data analyses.36.Export the Flow Cytometry Standard (FCS) data files.
Note: Voltages for FSC and SCC will depend on the cell line and cell treatment.
Note: Although the fluorophore of secondary antibodies defines the laser to be used, cytometer detector voltages can be different depending on the primary antibody.In this particular example, both Cy3 anti-rb and AF488 anti-ms control samples have been recorded 2 times, each one with the corresponding voltage of its primary antibody (MCM3 and STAG1 _ Cy3 / RAD21 and STAG2 _ AF488).That makes 8 compensation samples (Figure 5).
Note: Transient transfection of a POI before you begin (e.g., GFP-POI excited by B_525/20 laser) will require extra-gating.After step 33, gate positive cells from SSC-A vs B_525/20-H in order to make sure that 10,000 events are recorded for the population of interest.

Timing: 2.5 h
Flow cytometry data can be analyzed in multiple ways depending on the question.Quantitative analyses comparing the amounts of chromatin-bound proteins between different conditions (control and KD) and at specific cell cycle phases can be easily performed.For the example described here, we have used FlowJo software for analysis.However, alternative software such as FCS express or ModFit can be considered as well.
37. Drag and drop the FSC files and repeat gating strategy (steps 30-32).38.Export single FSC files for each population of barcoded samples.Make sure exports contain the same number of events (10,000 cells).See Figure 4.   43.Apply sample quality (green/blue: good quality; pink: poor quality in FlowJo).44.Use specific color compensation for each corresponding staining group.See Figure 5. 45.Create a layout.
a. Drag and drop your samples.b.Represent compensated-antibody-H vs.DNA content-A to observe the cell cycle profile of your protein of interest.c.Compare conditions among the same barcoded sample.See Figures 6A and 7.
Note: In steps 38-39, files can be exported in other formats than FCS3 (Figure 4), such as CSVscale values in order to allow for data quantification.See Figure 6B.

EXPECTED OUTCOMES
Staining and cell cycle analysis of MCM3 discloses successful extraction of soluble proteins.Although MCM3 profile shows a continuous increase from G1 to G2 when total proteins are analyzed (Figure 8 top), its chromatin-bound profile reveals that the protein loaded during G1 phase is progressively released by the passage of the replication fork during S phase (Figure 8 bottom).

LIMITATIONS
This method relies on the availability of high-quality antibodies that specifically recognize your POI and that work for flow cytometry.
Unlike immunofluorescence microscopy, Chromoflow does not provide information of the localization of the POI within the nuclear space.Treatments that affect protein distribution without changing total chromatin-bound levels will not show any difference in this assay.
The binding mode of your POI to DNA will dictate its sensitivity to the extraction method.Optimal extraction may require increasing the amount of salt in the extraction buffer, as suggested above.

3 .
Centrifuge (5 min at 0.1 RCF, 4 C) and resuspend cells in PBS (1 3 10 6 cells/mL).Keep your samples on ice.4. Centrifuge (5 min at 0.1 RCF, 4 C), remove all the supernatant and keep samples on ice.CRITICAL: Each tube should contain the same number of cells in order to ensure similar extraction for all conditions.

Figure 1 .
Figure 1.Sample preparation for extraction Each condition and staining tube contain 2 3 10 6 cells.Total number of cells per condition is 4 3 10 6 .12 3 10 6 cells (untreated or mock treated) are split in 6 control tubes.Image created with BioRender.com.

Timing: 45 min
This step will dye the nuclei/cells from each condition with a different concentration of Pacific Blue in order to multiplex the different conditions of your experiment in one single tube.13.Resuspend the fixed pellets in 195 mL PBS (FBS free).14.Transfer exactly 5 mL of the corresponding dye solution (L1, L2, L3, L4; see working solutions-Pacific Blue) into each tube (condition) and mix by pipetting up and down.See troubleshooting 2. 15.Incubate samples in the dark for 30 min at 25 C (RT).

Figure 2 .
Figure 2. Nuclear extraction Each centrifuge tube containing 2 3 10 6 cells is treated with 1 mL extraction buffer for 5 min in order to extract soluble nuclear components.Image created with BioRender.com.

Figure 4 .
Figure 4. Gating strategy for data analysis Gating strategy for uniform population (top left), single cell cycles (top middle) and different conditions in your experiment (top right) is required to export single FSC3 or CSV-scale values files (bottom).

Figure 5 .
Figure 5. Data compensation Specific compensation parameters (given by control samples) are applied to each staining group containing barcoded samples.

Figure 6 .
Figure 6.Data representation (A) Contour plots of exported FCS3 files.Compensated antibody of interest-H vs.DNA content-A is represented with adjacent histograms in a linear scale.Contour plots for STAG1, STAG2 and RAD21 in control (gray) and STAG1 KD, STAG2 KD or NIPBL KD conditions (colored) are overlapped for comparison.Data reanalyzed from Alonso-Gil et al. (2023).(B) Same data as A. Quantified median values from the exported CSV-scale values files were used to study log2 fold change (FC) enrichment of STAG1, STAG2 and RAD21 in all conditions.

Figure 7 .
Figure7.Versatile analyses Among other analyses, the behavior of your POI at specific moments of the cell cycle can be addressed.In this particular example, cell cycle representation as a DNA content-A histogram (left) allows to manually gate cells in G1 phase.Next, the amounts of STAG1 and STAG2 in these G1 cells can be plotted (right) and compared between control (gray) and KD conditions (colored).

Figure 8 .
Figure 8. Cell cycle profile of MCM3 Contour plots of exported FCS3 files.Compensated MCM3 antibody staining-H vs.DNA content-A is represented with adjacent histograms in a linear scale for total and chromatin association protocols (see optional step from nuclear extraction in step-by-step method details).Contour plots for MCM3 in control (gray) and STAG1 KD, STAG2 KD or NIPBL KD conditions (colored) are overlapped.

Table 2 .
Antibody staining strategy