Materials and reagents

Biological materials

Human peripheral mononucleated cells (PBMCs) are isolated from buffy coats using Ficoll Paque PLUS (Cytiva, catalog number: 17144002) according to the manufacturer’s instructions. Buffy coats from healthy human donors were obtained from the Australia Red Cross Blood Service with written and informed consent and with ethics approval from the University of Melbourne Human Research and Ethics Committee (#1035100).

Reagents

RPMI 1640 medium (Gibco, catalog number: 11875093)
Fetal bovine serum (FBS) (Gibco, catalog number: 5256701). Heat-inactivate at 56 °C for 30 min with interval mixing and store at -20 °C
Ethylenediaminetetraacetic acid-balanced salt solution (EDTA-BSS): 147 mM NaCl, 3.6 mM KCl, 0.01 mM KH₂PO₄, 0.02 mM K₂HPO₄, 14.5 mM HEPES, 6 mM EDTA (prepared in-house by dissolving each chemical in distilled water)
Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher, catalog number: 10010023)
10 mM sodium bicarbonate, pH 8.0, diluted from 1 M (Thermo Fisher Scientific, catalog number: J62495.AP)
Purified antibody specific for the cell surface protein to be assessed for internalisation or recycling (at a concentration of at least 2 mg/mL)
Cell type–specific surface markers for cell phenotyping, e.g.:
1. PerCP conjugated anti-CD3 (BD, catalog number: 345766)
2. BUV805 conjugated anti-CD14 (BD Horizon, catalog number: 612902)
Fixable viability dye eFluor780 (eBioscience, catalog number: 65-0865-14)
Click-iT^TM sDIBO Alkynes (Invitrogen, catalog number: C20025), dissolved in anhydrous DMSO at 1 mg/mL
Cy5-oligo-azide (5' Cy5-TCA GTT CAG GAC CCT CGG CT-N3 3') (Integrated DNA Technologies), dissolved at 150 μM in nuclease-free water and stored at -20 °C
Quencher (5' AGC CGA GGG TCC TGA ACT GA-BHQ2 3') (Integrated DNA Technologies), dissolved at 600 μM in nuclease-free water and stored at -20 °C
EZ-Link^TM Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific, catalog number: 21331)
Streptavidin-phycoerythrin (PE) (BioLegend, catalog number: 405203)
1 M Tris buffer, pH 8.0 (prepared in-house by dissolving Tris in distilled water and then adjusting pH with 1 M hydrochloric acid)
Sodium chloride (NaCl) (Spectrum Chemical, catalog number: SO160)
0.5 M EDTA, pH 8.0 (prepared in-house by stirring the required amount of EDTA powder in distilled water and then adjusting pH with solid sodium hydroxide pellets)
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906)
Sodium 2-mercaptoethanesulfonate (MESNa) (Sigma-Aldrich, catalog number: M1511)

Laboratory supplies

Zeba spin desalting column (7K, MWCO 0.5 mL) (Thermo Fisher Scientific, catalog number: 89882)
Amicon Ultra centrifugal filters (30 kDa, MWCO 0.5 mL) (Millipore, catalog number: UFC5030)
Low-protein binding 1.5 mL centrifuge tubes (Thermo Scientific, catalog number: 90410)
Round bottom 96-well plates (Corning, catalog number: 3799)

Solutions

Cell culture media (see Recipes)
Staining buffer (see Recipes)
Recycling assay buffer 1 (see Recipes)
Recycling assay buffer 2 (see Recipes)
MESNa incubation buffer (see Recipes)
Cell surface marker staining solution (see Recipes)

Recipes

Cell culture media (500 mL)
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
RPMI 1640 n/a 450 mL
FBS 10% (v/v) 50 mL
Total n/a 500 mL
Staining buffer (510 mL)
Note: Store at 4 °C.
Reagent Final concentration Quantity or Volume
EDTA-BSS n/a 500 mL
FBS 2% (v/v) 10 mL
Total n/a 510 mL
Recycling assay buffer 1 (500 mL)
Note: Dissolve the NaCl completely in milli-Q water before adding to the solution. Store at room temperature.
Reagent Final concentration Quantity or Volume
1 M Tris buffer, pH 8.0 50 mM 25 mL
NaCl 100 mM 2.9 g
0.5 M EDTA, pH 8.0 1 mM 1 mL
Milli-Q water Top up to 500 mL
Total n/a 500 mL
Recycling assay buffer 2 (50 mL)
Note: Prepare on the day of the assay. Scale down or up as required.
Reagent Final concentration Quantity or Volume
Recycling assay buffer 1 n/a 50 mL
BSA 0.2 % (w/v) 0.1 g
Total n/a 50 mL
MESNa incubation buffer (2 mL)
Note: Prepare on the day of the assay. Scale down or up as required.
Reagent Final concentration Quantity or Volume
Recycling assay buffer 2 n/a 2 mL
MESN 100 mM 0.033 g
Total n/a 2 mL
Cell surface marker staining solution
Note: Prepare on the day of the assay. Scale down or up as required. Replace with antibodies to differentiate your cells of interest.
Reagent Dilution Volume
PerCP conjugated anti-CD3 1:50 20 μL
BUV805 conjugated anti-CD14 1:200 5 μL
Fixable viability dye 1:1,000 1 μL
Staining buffer n/a 1,000 μL

Equipment

Air-cooled benchtop centrifuge (Beckman Coulter, model: Microfuge 22R Centrifuge)
Cell culture incubator at 37 °C and 5%–10% CO₂ (dependent on the recommended CO₂ level for your cells or media)
Flow cytometer (BD, model: LSR Fortessa FACS Calibur)

Software and datasets

FlowJo (version 10.4, 09/15/2017); can be purchased and downloaded online from https://www.flowjo.com/solutions/flowjo/downloads
Graph Pad Prism (version 10.1.0, 10/19/2023); can be purchased and downloaded online from https://www.graphpad.com

Procedure

Probe preparation

FIP
Overview: FIP is prepared by conjugating the antibody with Cy5-oligo using click chemistry of Click-iT^TM sDIBO Alkyne and the azide group.
1. Dilute 200 μg of antibody to 2 mg/mL in 10 mM sodium bicarbonate (pH 8.0).
2. Add 7 μL of Click-iT^TM sDIBO Alkyne and incubate for 2 h at 4 °C.
3. Remove the bottom closure of 7K Zeba spin desalting column and place it in a 1.5 mL tube.
4. Centrifuge the column at 1500× g for 1 min and discard the storage solution.
5. Add 300 μL of PBS to the column and centrifuge at 1500× g for 1 min. Discard flowthrough. Repeat twice.
6. Place the column in a 1.5 mL low-protein binding tube and add the protein-DIBO solution.
7. Centrifuge at 1500× g for 2 min and collect the eluate (containing the DIBO-labelled antibody eluate).
8. Add 20 μL of Cy5-oligo-azide to the eluate and incubate for at least 2 h (or ideally overnight) at 4 °C. This allows the reaction of conjugating the Cy5-oligo-azide to the DIBO-antibody forming the FIP.
9. Equilibrate the Amicon Ultra centrifugal filter by adding 500 μL of PBS and centrifuging at 13,000× g for 1 min. Discard flowthrough and any remaining PBS in the filter.
10. Top up the FIP solution (from step A8) to 500 μL in total with PBS.
11. Add to the Amicon Ultra centrifugal filter and centrifuge at 13,000× g for 5 min to remove excess unconjugated reagents. Discard flowthrough.
12. Repeat step A11 by rinsing the filter with 500 µL of PBS until the flowthrough is clear (approximately three washes).
13. Recover the purified FIP by placing the filter upside down in a new 2 mL tube and centrifuge at 1,000× g for 2 min.
14. Store the FIP in the dark at 4 °C for short-term use (<2 weeks) or aliquot and store at -20 °C for long-term storage.
15. FIP should be titrated to find the optimal staining dilution prior to beginning the assay as for normal flow cytometry antibodies (See Troubleshooting 2).
Recycling probe
Overview: Recycling probe is prepared by biotinylating antibody with EZ-Link^TM Sulfo-NHS-SS-Biotin that reacts with the primary amines on the antibody.
1. Dilute 200 μg of antibody to 2 mg/mL in PBS.
2. Prepare a 10 mM solution of Sulfo-NHS-SS-Biotin according to the manufacturer’s instructions.
3. Add 2.7 μL of Sulfo-NHS-SS-Biotin to the antibody (step B1).
4. Incubate the reaction on ice for 2 h to allow the conjugation of the Sulfo-NHS-SS-Biotin to the antibody.
5. Remove the bottom closure of 7K Zeba spin desalting column and place it in a 1.5 mL tube.
6. Centrifuge the column at 1,500× g for 1 min and discard the storage solution.
7. Add 300 μL of PBS, centrifuge at 1,500× g for 1 min, and discard flowthrough. Repeat twice.
8. Place column in a 1.5 mL low-protein binding tube and add the protein/Sulfo-NHS-SS-Biotin solution. Centrifuge at 1,500× g for 2 min and collect the antibody-SS-Biotin (the recycling probe) in the eluate.
9. Store the recycling probe in the dark at 4 °C for short-term use (<2 weeks) or aliquot and store at -20 °C for long-term storage.
10. Recycling probe should be titrated to find the optimal staining dilution prior to beginning the assay as for normal flow cytometry antibodies (See Troubleshooting 2).

Internalisation assay

Surface protein labelling
1. Prechill the centrifuge, plates, tubes, and solutions at 4 °C.
2. Add cells in 2 million cells per millilitre of suspension in cell culture media to a 1.5 mL centrifuge tube. Centrifuge at 620× g for 2 min. Wash cells once with staining buffer after removing supernatant.
3. Stain the cells with FIP diluted in staining buffer on ice for 20 min.
4. Wash the cells twice with ice-cold staining buffer.
5. Resuspend the cells in prechilled cell culture media at 2 million cells per millilitre.
Internalisation at 37 °C
1. Transfer 100 µL of the cell suspension (200,000 cells) into the appropriate wells of 96-round-bottom plates, on ice. Use two plates and perform each sample/time point in triplicate:
  1. Plate 1 (control plate, always on ice) requires two control samples:
    T_int = 0 –Q: Cells are not allowed to internalise and are not treated with quencher (Q).
    T_int = 0 +Q: Background control—cells are not allowed to internalise and are treated with Q.
  2. Plate 2 (internalisation plate, always in incubator at 37 °C).
    T_int = X +Q: Internalisation sample where cells are allowed to internalise for X time. Repeat for each time point.
2. Keep Plate 1 on ice. Incubate Plate 2 at 37 °C for the desired duration for internalisation to occur.
3. Transfer T_int = X +Q samples from Plate 2 to Plate 1 (on ice) at the end of each time point, terminating internalisation for that sample.
Quenching the un-internalised labelled protein and cell phenotyping surface marker staining
1. At this point, only Plate 1 remains. Wash the cells by adding 150 μL of ice-cold staining buffer to each well. Centrifuge the plate at 300× g for 5 min. Decant the supernatant. Repeat the wash.
2. Resuspend the cells in cell surface marker staining solution with or without Q (dilution 1/1,000) as indicated and incubate on ice for 15 min.
3. Wash twice with ice-cold staining buffer.
Measuring the percentage of internalised protein of interest
1. Run the cells on a flow cytometer.
2. Measure the geometric mean fluorescent intensity (gMFI) of the Cy5 (FIP) for each cell population using FlowJo (refer to Figure 2 for typical results). Calculate internalisation (see Data Analysis 1).
  
  Figure 2. Results from the internalisation or recycling assays for major histocompatibility complex (MHC) class I related protein 1 (MR1) using human peripheral blood mononucleated cells (PBMCs). (A) T cells and monocytes can be differentiated based on markers CD3 and CD14. The percentage of each population is shown in the plot. After identifying the cell population, (B) the fluorescence internalisation probe Cy5 signal (FIP-Cy5, using the antibody clone 8F2.F9 specific for MR1) for the internalisation assay or (C) the recycling probe–labelled Streptavidin-PE (using antibody clone 8F2.F9) for the recycling can be measured by flow cytometry. With the corresponding controls [T_int= 0 with and without quencher (Q) or MESNa cleavage (M)], the percentage of internalised or recycled molecules can be calculated. T_INT indicates internalisation time. T_REC indicates recycling time. Fluorescence minus one (FMO) indicates where the cells were stained with cell phenotyping antibodies and secondary staining but not FIP-Cy5 (B) or recycling probe (C). FMO served as the background control for the spread of signal in the multicolour panel and to know the quenching or cleaving effect in internalisation and recycling assays, respectively. Shown here for each assay, the typical results of one experiment (representing > 10) are shown by the geometric mean fluorescence (gMFI) and the calculated proportion of molecules that have been internalised (B) or recycled (C).

Recycling assay

Surface protein labelling
1. Prechill the centrifuge, plates, tubes, and solutions at 4 °C.
2. Add cells in 2 million cells per millilitre of suspension in cell culture media to a 1.5 mL centrifuge tube. Centrifuge at 620× g for 2 min. Wash cells once with staining buffer after removing the supernatant.
3. Stain the cells with staining buffer with the recycling probe on ice for 20 min.
4. Wash the cells twice with ice-cold staining buffer.
5. Resuspend the cells in prechilled cell culture media at 2 million cells per millilitre.
Internalisation at 37 °C
1. Transfer 100 µL of the cell suspension (200,000 cells) into the appropriate wells of 96-round-bottom plates, on ice. Use two plates and perform each sample/time point in triplicate:
  1. Plate 1 (control plate, always on ice) requires one control sample:
    –INT –M: Cells are not allowed to internalise (INT) and are not treated with MESNa (M).
  2. Plate 2 (internalisation/recycling plate, always in incubator at 37 °C except for Step C).
    +INT +M T_rec = 0: Background control—Cells are allowed to internalise, treated with M, but not allowed to recycle.
    +INT +M T_rec = X: Cells are allowed to internalise, treated with M, and then allowed to recycle for time X. Repeat for each time point.
2. Keep Plate 1 on ice. Incubate Plate 2 at 37 °C for the desired duration for internalisation to occur (typically 20–120 min). The optimal time for internalisation is the time required for a significant amount to be internalised—typically ~50%–100%; for MR1, this is 1 h (Figure 2B). This should be empirically tested for each molecule and cell type.
Cleaving biotin from the un-internalised labelled protein
Note: This step is conducted on all +M samples in plate 1 and 2 on ice.
1. Wash the +M samples once by pelleting down the cells by centrifuging at 300× g for 5 min and adding 200 µL of ice-cold recycling assay buffer 1 to the cell pellet in each well. Centrifuge again and decant the supernatant.
2. Resuspend the +M samples in 40 µL of ice-cold MESNA incubation buffer.
3. Incubate for 10 min on ice.
4. Wash the +M samples once with ice-cold recycling assay buffer 2.
5. Repeat steps C2–C4 once.
6. Wash once with culture media. Resuspend cells in culture media.
7. Transfer +INT +M T_rec = 0 samples from Plate 2 to Plate 1 (on ice) at the end of the internalisation and keep on ice.
Recycling at 37 °C
Note: At this point, only T_rec= X samples remain in Plate 2.
1. Incubate samples remaining in Plate 2 at 37 °C for time X, to allow recycling of the protein to occur.
2. At the end of each incubation, transfer the samples to Plate 1 on ice.
Staining of the recycled labelled protein and cell phenotyping surface marker staining
1. Wash all the samples twice with ice-cold staining buffer.
2. Resuspend the cells in cell phenotyping staining solution including Streptavidin-PE (1:200) and incubate on ice for 15 min.
3. Wash twice with ice-cold staining buffer.
Measuring the percentage of internalised protein of interest
1. Run the cells on flow cytometer.
2. Measure the gMFI of PE of each cell population using FlowJo (refer to Figure 2 for typical results). Calculate recycling (see Data Analysis 2).

Data analysis

Calculate the percentage of protein internalisation and recycling based on the ratio of the signal measured at the desired time point comparing to the total signal collected at time point 0 (when no internalisation/recycling occurs). Remove background signal in the calculation by subtracting the background control from these values.

Calculate the percentage of internalised protein at each internalisation time point (T_INT = X) as a fraction of the surface level before internalisation, subtracting the background signal (T_INT = 0+Q); where Q = quencher:
Calculate the percentage of recycled protein at each recycling time point (T_rec = X) as a fraction of the surface level before internalisation (–INT –M), subtracting the background signal (+INT +M T_rec = 0); where M = MESNa; INT = internalisation step:

Plot a time-course line graph using Graph Pad Prism if membrane protein internalisation or recycling are examined at multiple time points.

Validation of protocol

This protocol or parts of it has been used and validated in the following research article(s):

Lim et al. [15] “A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells”, (Journal of Cell Biology), Figure 1A-C, 2D-E, 2H, 3F-G, 7B-C. Performed on human PBMC, human B lymphoblast cells line C1R cells and human monocyte cell line THP-1 cells.
McWilliam et al. [16] “The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1” (Nature Immunology), Figure 5A. Performed on human C1R cell line.
Howson et al. [17] “Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1” (Science Immunology), Figure 5C. Performed on human C1R cell line.

General notes and troubleshooting

General notes

These methods can be adapted to study different surface proteins in suspension cells from different hosts.
These methods are not suitable for adherent cell lines. Adherent cells may attach to the plate during the incubation at 37 °C. Trypsin treatment for the cell lifting may alter the cell behaviour and cell membrane biology, leading to inaccurate measurement in the assay.
The choices of fluorophores mentioned are those used by our studies; however, other fluorophores can also be used.
The recycling assay here measures the percentage of the originally labelled surface molecules that are recycled back to the cell surface after a period of internalisation. For the interpretation of the results, it is important to keep in mind that this rate is dependent on both the rate of recycling and the length of time used for the internalisation step. Hence, the length of time used for the internalisation step should be reported for this assay.
It has not been determined if fixation affects the performance of these assays. It is possible that fixing the cells after the assay is compatible with accurate measurements of internalisation and recycling, but this should be verified first by comparing the results of fixed vs. unfixed cells.
The protocol here uses the gMFI of the probes to measure the internalisation or recycling rate for the whole population of interest. It is possible, however, to use other measures or calculations distinct from gMFI to determine if there are heterogenous rates of internalisation within each population.

Troubleshooting

Problem 1: Too few cells acquired during flow cytometry.

Possible cause: Cells are lost during the washing steps, or the assay started with a low number of cells.

Solution: Increase the number of cells used in the assay.

Problem 2: Signal from positive controls (–Q or –M) is low.

Possible cause: The probes (FIP or recycling) are too diluted during the staining, or the expression of the protein being studied has a low abundance.

Solution: Optimise the concentration of the probe for the staining, until a stronger signal is gained. Use the best dilution of probes that give best signal (–Q or –M) to background (+Q or +M) ratio.

Problem 3: Inconsistent results.

Possible cause: Plates used for storing samples or controls are not kept cold and increase in temperature, hence internalisation or recycling occurs during washing or staining.

Solution: Use a prechilled centrifuge, consumables, and solutions throughout the assay unless specified to prevent internalisation and recycling after the time points have ended.

Problem 4: No recycled molecule detected in recycling assay.

Possible cause: Insufficient or extended time for the internalisation to occur.

Solution: Adjust the duration for internalisation at 37 °C in the way that the gMFI of +INT –M T_rec = 0 is a lower signal than the gMFI of –INT –M, yet higher than that of +INT +M T_rec = 0.

Acknowledgments

The work was supported by a National Health and Medical Research Council (NHMRC) Ideas Grant (2003192). The internalisation assay detailed in this protocol was adapted from the internalisation method developed by Liu et al. [1,2]. The recycling assay was adapted for flow cytometry from a biochemical assay previously used [12,13]. We acknowledge the pioneering work of these authors.

Competing interests

The authors declare no competing interest.

Ethical considerations

Human blood from healthy human donors was obtained from the Australia Red Cross Blood Service with written and informed consent and with ethics approval from the University of Melbourne Human Research and Ethics Committee (#1035100).

References

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Lim, H. J., Wubben, J. M., Garcia, C. P., Cruz-Gomez, S., Deng, J., Mak, J. Y. W., Hachani, A., Anderson, R. J., Painter, G. F., Goyette, J., et al. (2022). A specialized tyrosine-based endocytosis signal in MR1 controls antigen presentation to MAIT cells. J. Cell Biol. 221(12). https://doi.org/10.1083/jcb.202110125.
McWilliam, H. E. G., Eckle, S. B. G., Theodossis, A., Liu, L., Chen, Z., Wubben, J. M., Fairlie, D. P., Strugnell, R. A., Mintern, J. D., McCluskey, J., et al. (2016). The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1. Nat. Immunol. 17(5): 531–537. https://doi.org/10.1038/ni.3416.
Howson, L. J., Awad, W., von Borstel, A., Lim, H. J., McWilliam, H. E. G., Sandoval-Romero, M. L., Majumdar, S., Hamzeh, A. R., Andrews, T. D., McDermott, D. H., et al. (2020). Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1. Sci. Immunol. 5(49). https://doi.org/10.1126/sciimmunol.abc9492.