Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling

Summary Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).1

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Highlights O-ClickFC allows flow cytometric analysis of PC metabolism in live mammalian cells
High-throughput measurements of PC at the organelle level with single-cell resolution A simple and easy-touse protocol using commercially available reagents

MATERIALS AND EQUIPMENT
Note: All media are stored at 4 C up to 2 months.

Reagent
Final concentration Volume (mL)

STEP-BY-STEP METHOD DETAILS Metabolic incorporation of N 3 -Cho
Timing: 1 h This section describes the metabolic incorporation of N 3 -Cho into PC in the K562 cells (Figure 1).
Note: All steps are performed at room temperature otherwise indicated.
CRITICAL: Wash the cells with choline-free medium 2-3 times to completely remove choline residues.
7. Cell count with automated cell counter.8. Adjust cell density to 0.5-1.0 3 10 6 cells/mL with Cho-free medium.9. Add 10 mM N 3 -Cho to final concentration of 10 mM.Mix well.10.Inoculate the cell suspension into 12-well plate (1 mL/well).11.Culture cells in humidified incubator at 37 C, 5% CO 2 for 16-24 h.Note: At this point, the protocols diverge depending on the target of the interest (ER-Golgi, Plasma membrane or mitochondria).Choose one of the methods below to continue the protocol at step 12 for ER-Golgi, step 22 for plasma membrane, or step 33 for mitochondria.

Organelle-specific labeling of N 3 -PC Labeling of N 3 -PC in the ER-Golgi
Timing: 45 min This section describes the fluorescence labeling of N 3 -PC using the ER-Golgi-targeting clickable dye BDP-DBCO (Figure 1).Note: 12-21 steps are performed at room temperature.
Optional: 1 mL of FVD780 can be added to assess cell viability.
CRITICAL: It is important to make cells dispersed during click chemistry reaction.
Note: Fluorophore used in this study is relatively stable, and thus filtered cells can be stored at room temperature for up to 8 h without reducing the fluorescence signals.However, it is recommended to analyze the cells as quickly as possible.

Timing: 1 h
This section describes the fluorescence labeling of N 3 -PC using the plasma membrane-targeting clickable dye DBCO-AF647 (Figure 1).Optional: 1 mL of FVD780 can be added to assess cell viability.
CRITICAL: It is important to make cells dispersed during click chemistry reaction.
Note: Fluorophore used in this study is relatively stable, and thus filtered cells can be stored at room temperature for up to 8 h without reducing the fluorescence signals.However, it is recommended to analyze the cells as quickly as possible.

Protocol
Labeling of N 3 -PC in mitochondria Timing: 2 h This section describes the fluorescence labeling of N 3 -PC using the mitochondria-targeting clickable dye Cy3-DBCO (Figure 1).Optional: 1 mL of FVD780 can be added to assess cell viability.
CRITICAL: It is important to make cells dispersed during click chemistry reaction.
Note: Fluorophore used in this study is relatively stable, and thus filtered cells can be stored at room temperature for up to 8 h without reducing the fluorescence signals.However, it is recommended to analyze the cells as quickly as possible.

Timing: 1 h
This section describes the gating strategy for optimal data acquisition (Figure 2).50.Optimize the flow cytometry instrument setting for optimal data acquisition.If using MA900 (Sony), following parameter can be used as a reference.
51. Scatter density plot (FSC-A vs. BSC-A, all events) (Figure 2A): Set up a plot for FSC vs. BSC area to identify cells of interest.
Note: For K562 cells, typically live cells lie within the range of 20-70 (310,000) in FSC-A and 10-40 (310,000) in BSC-A (Gate A).Events with <20 (310,000) in FSC-A are either debris or dead cells and should be gated out.Any events that lie >70 (310,000) in FSC-A and >40 (3 10,000) in BSC-A are likely non-singlets and should be avoided.
Note: Doublet cells can significantly affect your analysis and could lead to inaccurate conclusion.Gate for an area where majority (90%-100%) of events reside (Gate B).
Optional: Live cells can be selected by FVD780-negative staining (Figure 2C).

EXPECTED OUTCOMES
Using the above methods, N 3 -Cho treated cells will display higher fluorescence intensity in labeled PC than untreated cells (Figure 2D).The median fluorescence intensity (MFI) of the PC labeling differs approximately 10-to 100-fold between N 3 -Cho-treated cells and untreated cells (Figure 2D).This method can be combined with CRISPR-Cas9 knockout system or pharmacological experiments to study PC metabolism.Administration of N 3 -Cho into mice should allow PC labeling of cells isolated from tissues and fluids.This method is compatible with conventional cell surface immunofluorescence of live cells using fluorophore-conjugated antibodies for cell lineage identification, but not compatible with intracellular staining.

LIMITATIONS
The protocol shown here is based on floating K562 cells, and therefore appropriate optimization for adherent cells should be required.FBS concentration is critical for avoiding non-specific binding of dyes to cells as well as removing un-reacted dyes.When researchers do not use FBS, this protocol Protocol cannot be used.In terms of experimental temperatures, the plasma membrane labeling needs to be conducted below 15 C, in order to minimize endocytosis.The mitochondrial labeling depends on the mitochondrial membrane potential which is maintained at 37 C. Overall, the uncontrolled temperatures potentially affect organelle-selective localization of the dyes and the labeling efficiency in the targeted organelles, which might lead to undesirable results such as nonspecific adsorption of dyes.Thus, adherence to temperature control guidelines in this protocol is critical for achieving organelle selectivity in PC labeling.

TROUBLESHOOTING Problem 1
Low or no fluorescence in labeling of PC (related to metabolic incorporation of N3-Cho and organelle-specific labeling of N3-PC).

Potential solution
This may occur when low or no N 3 -Cho has been incorporated into the cells (step 9), or incorrect concentration of clickable dyes was used (steps 17, 27, or 38).For the former, any of choline remained in the culturing medium should be removed prior to the N 3 -Cho incubation, as choline is a competitive inhibitor of N 3 -Cho.Wash the cells with choline-free medium at least twice, or increase the final concentration of N 3 -Cho.For the later, check the concentration of the clickable dye use (especially for the plasma membrane labeling, which needs higher concentration of the dye than the other labeling).To gain sufficient fluorescence signals in labeling, an optimal ratio of dye amount, cell number and medium volume during click reaction should be determined.

Potential solution
As described in Limitation section, organelle selectivity is sensitive to temperature.Check and control instrumental temperature and incubation time (steps 17, 27, or 38), if unintended staining of dyes is observed.

Problem 3
Broad or heterogeneous populations in PC labeling (related to flow cytometry).

Potential solution
This may occur when DBCO reagents and cells are unevenly distributed (steps 17, 27, or 38).Resuspend cells with DBCO working solution and mix well (check the CRITICAL points in organelle-specific labeling of N3-PC).

Problem 4
Toxicity in mitochondrial washing steps (related to labeling of N3-PC in mitochondria).

Potential solution
Loss of mitochondrial membrane potential (step 42) promotes removal of unreacted Cy3-DBCO, but the excessive treatment with High K+ washing medium potentially causes cell damage.If obvious toxicity is detected, reduce concentration of mesoxalonitrile 3-chlorophenylhydrazone and valinomycin, or shorten incubation time using High K+ washing medium.

Problem 5
Altered culture conditions (related to metabolic incorporation of N3-Cho).

Potential solution
If a medium different from this protocol is used (steps 4-11), efficiency in N 3 -Cho incorporation may be changed.In this case, optimize amount of N3-Cho in cell culture.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Itaru Hamachi (ihamachi@sbchem.kyoto-u.ac.jp).

Materials availability
This study did not generate new unique reagents.

Figure 1 .
Figure 1.Scheme for metabolic N 3 -Cho incorporation and fluorescent PC labeling Steps 1-11 for metabolic incorporation of N 3 -Cho into PC in K562 cells.Steps 12-21 for PC labeling in ER-Golgi using BDP-DBCO.Steps 22-32 for PC labeling in the plasma membrane using DBCO-AF647.Steps 33-49 for PC labeling in mitochondria using Cy3-DBCO.
53. Analysis of fluorescent-labelled PC (Fluorescence vs. Events, gate C) (Figure 2D): set up a histogram plot for each organelle-selectively fluorescent-labelled PC to analyze its distribution.a.For analyzing BDP-labeled PC, set up a histogram plot in the green fluorescence channel (FL1 vs. Events for MA900; Figure 2D middle).b.For analyzing abundance of AF647-labeled PC, set up a histogram plot in the far-red fluorescence channel (FL10 vs. Events for MA900; Figure 2D top).c.For analyzing Cy3-labeled PC, set up a histogram plot in the orange fluorescence channel (FL2 vs. Events for MA900; Figure 2D bottom).
High K + washing medium (working) should be prepared fresh during the procedure and stored at 37 C until use.