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Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells

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Scientific Reports
Feb 2016


Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.

Keywords: Influx transporters (流入转运蛋白), Anthracyclines (蒽环类药物), Autofluorescent drugs (自体荧光药), FACS analysis (FACS分析), Epifluorescence microscopy (荧光显微镜检查), Fluoroskan reader (Fluoroskan读数仪)


The anthracyclines, such as doxorubicin and daunorubicin, act by damaging the DNA and are used for treating various types of cancers including acute myeloid leukemia. When cancer patients are given anthracyclines systemically, there are several factors limiting the amount of the drugs that reach the tumor sites (Chauncey, 2001; Deng et al., 2014; Riganti et al., 2015). In the tumor, the drug activity on the cancer cells is limited by poor drug uptake, excessive drug efflux as well as changes in the cellular targets. For several decades, it remains unclear how these DNA damaging agents enter cancer cells (Aouida et al., 2010; Cesar-Razquin et al., 2015; Zhang et al., 2015). To address this question, we developed three reliable in vitro assays to monitor daunorubicin accumulation into cells. Two of these assays are quantitative and required access to a Fluorescence-Activated Cell Sorting (FACS) caliber and a Fluoroskan instrumentation and the third is semi-quantitative using epifluorescence microscopy. Using these assays, we established that daunorubicin enter into cells in a time- and concentration-dependent manner and that each cell type showed a different rate of uptake, suggesting that an active process is involved in the uptake of anthracyclines (Andreev et al., 2016). We applied these assays and uncovered the organic cation transporter 1 (OCT1) as a key protein for the uptake of daunorubicin into the cells (Andreev et al., 2016). Modulating the level of OCT1 resulting in cells with altered uptake and sensitivity towards daunorubicin (Andreev et al., 2016). These assays provide hints that additional transporters exist to allow uptake of daunorubicin into the cells. We believe that these uptake assays can be exploited further to identify additional factors such as kinases (Tanaka et al., 2004; Ciarimboli and Schlatter, 2005; Zhou et al., 2005; Pelis et al., 2006; Filippo et al., 2011; Sprowl et al., 2016) that influence the rate of daunorubicin uptake. In this protocol, we describe three analyses to monitor the uptake of anthracyclines into cells.

Materials and Reagents

  1. Mammalian cell culture containers for adherent cells (100 and 60 mm Petri dishes and T-75 flask)
    100 mm Petri dishes (SARSTEDT, catalog number: 83.3902 )
    60 mm Petri dishes (SARSTEDT, catalog number: 83.3901 )
    T-75 flask (Corning, catalog number: 430641 )
    1. However, any other Petri dishes or flask suitable for your cell line may work.
    2. Note that you can grow suspension cells in containers for adherent cells.
  2. Eppendorf tubes
  3. FACS tubes that will adapt to the flow cytometer:
    Falcon 5 ml polystyrene round-bottom tube (Corning, Falcon®, catalog number: 352058 )
  4. Microscope slides (UltiDent Scientific, catalog number: 170-7107A )
    Note: Any microscope slide that fit on your microscope can be used.
  5. Cover glass (UltiDent Scientific, catalog number: 170-C1818 )
    Note: Any cover glass can be used. The best cover glasses are the #1.5, however #1.0 or #0 may be used if high quality images are not required (e.g., confocal microscopy).
  6. Black clear flat bottom 96-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 165305 )
  7. Mammalian adherent cells, for example, HeLa, HEK293 and TOV112D
    Note: Suspension cells such as HL60 and K562 can be used, although the protocol would need to be adjusted.
  8. Required complete culture media
    1. DMEM (WISENT, catalog number: 319-005-EL )
    2. 1x RPMI 1640 (WISENT, catalog number: 350-000-CL )
    3. Ovarian Surface Epithelial (OSE) (WISENT, catalog number: 316-030-CL )
    Note: We used the above growth media depending on the cell line. Verify with the cell line suppliers to determine the optimal growth medium for the cells and the necessary supplements.
    1. Fetal bovine serum (FBS) (WISENT, catalog number: 095150 )
      Note: It is usually used at 10%, but some cell lines will require different concentrations. See with your cell line supplier.
    2. Penicillin/streptomycin (WISENT, catalog number: 450-201-EL )
      Note: It is usually supplied as a 100x stock and therefore must be diluted 1:100 in DMEM and RPMI1640.
    3. Gentamycin Sulfate 50 mg/ml Solution (WISENT, catalog number: 450-135-XL ) supplied at 1,000x and must be diluted 1:1,000 in the media
    4. Amphotericin B (Sigma-Aldrich, catalog number: A4888 ) used at 0.5 μg/ml in OSE
      Note: We usually prepare a stock solution of 250 μg/ml and add 1 ml of to 500 ml of OSE.
  9. Trypsin (0.05% or 0.25% in PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  10. Daunorubicin
    1. Provided by the pharmacy (Maisonneuve-Rosemont Hospital), but can be purchased from Sigma (Sigma-Aldrich, catalog number: 30450 ). Prepared at 5 mg/ml in sterile water to a final molar concentration of 8.87 mM.
    2. Daunorubicin may be replaced by doxorubicin (Sigma-Aldrich, catalog number: D1515 ).
  11. 4% paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 ) solution in PBS
  12. Mounting medium with DAPI (Vectashield, Vector Laboratories, catalog number: H-1200 )
    Note: This component may be replaced by 50% glycerol (Bio Basic, catalog number: GB0232 ) containing 1 to 10 μg/ml Hoechst 33342 (Thermo Fisher Scientific, InvitrogenTM, catalog number: H1399 ).
  13. Nail polish
  14. Phosphate buffer saline (PBS) (see Recipes)
    Sodium chloride (NaCl) (WISENT, catalog number: 600-082-IK )
    Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
    Sodium phosphate dibasic (Na2HPO4) (Bio Basic, catalog number: S0404 )
    Potassium phosphate monobasic (KH2PO4) (Bio Basic, catalog number: PB0445 )
  15. Uptake buffer (see Recipes)
    Sodium chloride (NaCl) (WISENT, catalog number: 600-082-IK )
    HEPES pH 7.4 (Bio Basic, catalog number: HB0265 )
    Glucose (WISENT, catalog number: 600-350-IK )
    Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P4504 )
    Potassium phosphate monobasic (KH2PO4) (Bio Basic, catalog number: PB0445 )
    Calcium chloride (CaCl2) (Fisher Scientific, catalog number: C79-500 )
    Magnesium sulfate (MgSO4) (Bio Basic, catalog number: MN1988 )


  1. Pipettes (any pipettes will do)
  2. Incubator (any incubator is fine if it maintains 5% CO2 and moisture)
  3. pH meter (any pH meter will work)
  4. Eppendorf centrifuge (any centrifuge that can adapt 1.5 ml microcentrifuge tube)
  5. Hemocytometer (any hemocytometer will do)
  6. Flow cytometer (FACS)
    Note: This protocol is using a FACS Calibur from Becton-Dickson (BD, model: FACS CaliburTM ).
  7. Epifluorescence microscope (Olympus, model: BX53 )
    Note: We use an Olympus BX53 , but any epifluorescence microscope with the required filter sets may be used (see in Procedure D for the required filters).
  8. Fluoroskan Ascent (Thermo Fisher Scientific, Thermo ScientificTM, model: Fluoroskan AscentTM )


  1. ImageJ
    Note: We used the one at the National institute of Health website; https://imagej.nih.gov/ij/.
  2. CellQuest Pro either version 4.0.1 © 1994-2001 BDB or 5.2.1 © 1994-2005 BD


  1. Preparation of the cells
    1. Grow the cells of interest in the recommended culture media at 37 °C, 5% CO2, 95% air.
    2. The day before the experiment, remove media from the cells and wash with PBS (see Recipes). Add appropriate volume of trypsin (1-2 ml for 100 mm Petri dish or T-75 flask). Incubate at 37 °C for 2 to 15 min.
      Note: The incubation time varies between cell lines. Incubate until you can see the cells detaching from the surface.
    3. Add complete media to the cells to inhibit the trypsin.
      Note: The volume of media to add to the cells depends on the number of samples to be created. Keep in mind that you have to divide this volume by the number of samples you want to create.
    4. Pipette the cells into 60 mm Petri dishes with a total of about 5 to 6 ml of media.
      1. The number of plates needed depends on the kind of experiment you want to perform. You always need at least one Petri plate for negative control, but it is better to have all samples in triplicate. You will need three plates for each time point or three plates for each daunorubicin concentrations to be tested.
      2. You must seed your cells so the confluence on the day of the experiment is below 50% as higher confluence will artificially reduce daunorubicin uptake.
    5. Incubate cells at 37 °C, 5% CO2, 95% air.

  2. Treatment of the cells with daunorubicin
    1. Remove the culture media from the cells.
      Note: Cells can be washed, but this does not seem to affect the overall results. However, if cells were treated with additional compounds prior to daunorubicin treatment, a washing step with UB should be added.
    2. Add 5 ml of UB to the cells.
    3. Incubate at 37 °C, 5% CO2, 95% air for at least 5 min.
    4. Prepare a daunorubicin dilution of 255 µM in UB by diluting the stock concentration of 8.87 mM by 35-fold.
    5. Add 100 µl of 255 µM daunorubicin to the 5 ml of UB to have a final concentration of 5 μM.
      1. To test different concentrations of daunorubicin, add the required volume of diluted daunorubicin keeping in mind to adjust the volume of UB added to the cells before. Trying various concentrations may be a good thing in order to determine the best concentration for your experiment. The reference concentration should be 5 µM.
      2. Keep in mind that you will need an untreated cell sample for all the manipulations.
    6. Incubate the cells at 37 °C, 5% CO2, 95% air for the required time.
      Note: Daunorubicin uptake can be detected after a few minutes, but longer exposure time may increase the difference between samples. Therefore, a time-dependent curve may be necessary in order to determine the best time point for your experiment. An exposure time between 30 to 60 min would be the best starting point.
    7. Once the incubation at 37 °C is finished, depending on the cell type they may start to detach from the plate. If this is the case, collect the cells by up and down pipetting to detach all the cells and put them in Eppendorf tubes. Pellet the cells at 5,000 x g for 1 min using any standard Eppendorf centrifuge and remove the supernatant. Wash the cells with 1 ml of cold PBS.
      Note: If the cells did not detach from the plate, remove the UB containing the daunorubicin and wash the cells with cold PBS. Add 0.5 ml of trypsin and wait for the cells to detach. Add 0.5 ml of cold PBS and collect all the cells in an Eppendorf tube and centrifuge at 5,000 x g for 1 min. Remove supernatant.
    8. To the cell pellets, add 100 µl of 4% PFA and incubate for 10 min at room temperature or 60 min at 4 °C.
    9. Centrifuge 5,000 x g for 1 min and remove the supernatant.
    10. Add 100 µl of PBS.
      Note: From this point, you can go to either Procedure C (FACS analysis), Procedure D (Epifluorescence Microscopy) or Procedure E (Fluoroskan analysis).

  3. FACS analysis of the treated cells (need roughly 100,000 cells for the analysis)
    1. Take one FACS tube for each sample you have and put 250-500 µl of PBS in each tube.
      Note: The amount of PBS you put in the tube depends on the number of cells you have. More PBS you put, more time it will take to reach the required number of events on the FACS. However, you need sufficient volume to make your adjustment on the machine.
    2. Add the cells prepared in Procedure B to the PBS in the FACS tube.
      Note: You can always keep some cell suspension to perform epifluorescence microscopy and Fluoroskan analyses as in Procedure D and Procedure E, respectively, but this would depend on the size of the cell pellets in Procedure B. Keep in mind that it will take longer to read 10,000 events if there are less cells in the FACS tube.
    3. Turn on the FACS and the computer and make sure the valve is closed so the FACS Flow tank is under pressure.
      The tanks are in the ‘drawer’ of the machines. There are two tanks, the right one is the FACS Flow, the left one is for the wastes. The valve is at the back between the tanks.
    4. Open the software of the FACS (here we are using CellQuest Pro).
    5. Go to ‘Acquire’ tab and click on ‘Connect to cytometer’ (Figure 1).

      Figure 1. Showing the display to connect the cytometer to the computer

    6. Enter the name for each needed detector. Use P4 (FL2 585/42) for daunorubicin detection (Figure 2).

      Figure 2. Displaying the Browser. Enter names for each used detector.

    7. Choose the folder where you want to save your data by clicking on the first ‘Change’ (Figure 3).

      Figure 3. Displaying within the Browser, the Directory. Selection of the folder that will contain all the file of the experiment.

    8. Draw a dot plot or a density plot and change the ‘Plot Type’ to acquire˃˃analysis (Figure 4).

      Figure 4. Plot creation. Make sure to change the plot type.

    9. Click on ‘Edit’ tab and ‘Duplicate’ to create another graph. On the second graph, change the x axis name to daunorubicin.
      Note: This is like doing ‘copy/paste’, but it doesn’t work to duplicate graph in CellQuest Pro and this is why one must use ‘duplicate’ function. Graphs could also be created one by one, but this requires changing the ‘plot type’ each time.
    10. In the ‘Cytometer’ tab, click on ‘Detector/Amps’. Change the fluorescent one to ‘Log’ mode (Figure 5).

      Figure 5. Showing the detector controls. Change the scale type and adjust voltage of detectors.

    11. Click on the quadrant tool and create quadrants in the daunorubicin graph placing the vertical line around 10 and the horizontal line around the middle of the graph (Figure 6).
      These will constitute thresholds. You can change these settings according to your sample if required.

      Figure 6. Showing the quadrant. The thresholds of your experiment.

    12. Click on the ‘Stats’ tab and ‘Quadrant Stats’ (Figure 7).
      You can edit the quadrant stats if you go back in the ‘Stats’ tab (Figure 7).

      Figure 7. Display and edit quadrant stats

    13. Remove any tube from the machine and press ‘Prime’. Once the light of the button is turned off, press again.
    14. Place one of your negative control tubes on the machine and press ‘Run’.
    15. Ensure the ‘Setup’ check box is checked and click on ‘Acquire’ (Figure 8).

      Figure 8. The Browser in ‘Setup’ mode. This allows you to analyze cells without saving any data.

    16. Change the voltage of the detectors to have almost all the cells in the lower left quadrant. (Figure 9)
      1. Using SSC as y axis, place the horizontal line of the quadrants such as to exclude cells with a very high SSC value which have high chances of being dead cells. Thus, the upper quadrants will exclude the dead cells.
      2. Another option to exclude dead cells from the analysis is to create a gate on the graph displaying FSC and SSC of the cells (P1 and P2). Dead cells will have a very low FSC and a high SSC. To do so, on the FSC-SSC graph, use the gate tools to select the living cell population. In the menu of the daunorubicin graph, go to ‘Gate’ and select ‘R1’ or the appropriate gate.

      Figure 9. Adjust detectors voltage

    17. Click on the ‘Set Up’ check box to remove the check and write the name of your first sample in the sample text box (Figure 10).
      You can also use the patient text box to write part of the name that will not change often. As an example, if you are doing time dependent curve with two cell lines, write the cell line’s name in the patient text box and the time point in the sample text box (Figure 10).

      Figure 10. Preparing the acquisition

    18. Place the sample to be analyzed on the machine and click on ‘Acquire’ (Figure 11).
      You can see the number of events per second and the total number of events by displaying the ‘Counters’ window in the ‘Acquire’ tab. The speed of the machine can be changed from slow to medium to high directly on the machine. Having 1,000 events per second is the best.

      Figure 11. Acquisition

    19. Change your tube and repeat the acquisition for all your samples.
    20. Once you have analyzed all your samples, you can display them in the graph by clicking on the graph and then, in the ‘Plot’ tab you can change data displayed (Figure 12).

      Figure 12. Navigation between samples

    21. To analyze the data, multiply the mean daunorubicin fluorescence in the lower right quadrant by the percentage of cells in that quadrant displayed in the quadrant stats window (Figure 13).

      Figure 13. Calculation of the average fluorescence of the sample

      Note: If you are using cell line transfected with a fluorescent marker, for example, studying the effect of overexpression of a particular protein tagged with GFP, create another graph and change the x axis of that graph for P3 (FL1 530/30). Create a gate using the gating tools to select only the GFP positive cells using a non-transfected cell sample as negative control to set the threshold. Next, within the menu of the graph that displays the daunorubicin fluorescence, go to Gate and select R1. This way, the graph displaying the daunorubicin will only display GFP positive cells. This can be very useful if you are doing transient transfection. However, you might need to increase the number of events to get enough GFP positive cells in the daunorubicin graph. To do so, go in the Acquire tab and click on acquisition and storage. There you can set the even count. There could be GFP leak on the FL2 detector, as such using P5 (FL3) may be an option. You can also use the compensation menu in the cytometer tab to reduce the effect of the GFP on the FL2 and the effect of the daunorubicin on the F1.

  4. Epifluorescence microscopy
    1. Put 3 μl of mounting medium with DAPI on a microscope slide.
      Note: Mounting medium with DAPI can be replaced by 50% glycerol solution containing 1 to 10 μg/ml of Hoechst 33342.
    2. Add 2 μl of cell suspension from Procedure B.
    3. Put a cover slip on the sample and fix it with nail polish.
      Note: Slides can be stored at 4 °C in the dark for one to two weeks.
    4. Visualize the cell with an epifluorescence microscope using DAPI filter to visualize nucleus and Red filter (Texas RED) to visualize daunorubicin at 60x or 100x.
      Note: The protocol here describes the procedure for an Olympus B53 upright epifluorescence microscope working on Cellsens.
      1. DAPI filter is working also for staining with Hoechst 33342.
      2. To visualize daunorubicin you must excite with a wavelength going from 480 nm to 560 nm. Excitation through a FITC or Texas RED filter should work. The emission of daunorubicin can be detected from 575 nm to 680 nm (Texas RED/mCHerry filter). Using a Texas RED filter set would be the best, otherwise different filters for emission and excitation could be required.
    5. Using a control sample, set the light intensity and the exposure time so the daunorubicin is clearly visible, but not saturated (Figure 14).

      Figure 14. Example of images to be obtained

    6. To adjust the light intensity, use lamp device connected to the microscope.
    7. The adjustment of the exposure time is done on the computer in the ‘exposure window’ (Figure 15).
      To use this method in a quantitative way you must not change these parameters for the daunorubicin visualization.

      Figure 15. Adjusting the exposure time

    8. Adjust these parameters (light intensity and exposure time) also for nucleus.
      These settings can be changed from one sample to another unless you want to study the chromatin itself. The staining of the chromatin by DAPI or Hoechst can be compromised by high amount of DNR onto the DNA.
    9. If using GFP or another color, set the light intensity and exposure time for this color as well.
    10. Take pictures of all samples for all colors. To do so, you must click on ‘Snap’ (Figure 16).

      Figure 16. Camera controls. Snap to take a picture.

    11. You can navigate between the pictures you took by clicking on the tabs above the pictures (Figure 17).
      1. The ‘live’ tab allows you to see live what is on the microscope. To display it, if it is not, click on the live button.
      2. Important Note: Microscopes have ‘modes’ so they display through the ocular, the camera or both. When you take picture, make sure the ‘mode’ is one that is using the camera. On Olympus B53, this can be by pulling or pushing a metal stick on the right side of the oculars.
      3. Some microscopes such as ZEISS Z2 use special software that can also serve to edit the pictures to create TIFF files with colors and a merge pictures.

      Figure 17. Navigation between the pictures

    12. Save all the images which can then be processed with ImageJ.
    13. Open the ImageJ software and click on file > to open the images. Open all the colors of a single sample (Figure 18).

      Figure 18. Open pictures in ‘File’

    14. Click on the picture, then go to the ‘image’ > channels tool (Figure 19).

      Figure 19. Open the ‘Channels Tool’

    15. On the channel window, click on ‘more’ and then click on the required color for your selected picture. Apply the colors to all pictures (Figure 20).

      Figure 20. Apply the colors to your pictures

    16. Click on ‘Merge Channels’ and select the required picture for each color. Click ‘OK’ (Figure 21).
      If you do not want the software to close all your opened pictures, make sure the ‘Keep source images’ check box is checked (Figure 21).
    17. You can now ‘Save as’ all pictures as desired file type: TIFF, JPEG, etc.

      Figure 21. Merge your images

  5. Fluoroskan analysis
    1. Dilute the cell suspension from Procedure B 1:10 to 1:100 depending on the cell density.
    2. Put 10 μl of the dilution on a hemocytometer and count the number of cells in the 25 central squares and multiply the number by 20,000.
    3. Then multiply the number obtained by the dilution factor to obtain the number of cells per ml in the cell suspension.
      Note: Do this for each sample to be analyzed.
    4. In a black clear flat bottom 96-well plate, put 6,000 to 20,000 cells and complete the volume in each well to 100 μl with PBS. Each well must have the same amount of cells.
      Note: It is recommended to do analyze each sample in triplicate.
    5. Put the plate on the Fluoroskan plate reader.
      Note: Make sure to remove the cover of the 96-well plate.
    6. Click on ‘Measure1’ (Figure 22).

      Click on ‘Measure1’ (Figure 22).

      Figure 22. Open the ‘Measure1’ window

    7. Select the wavelength pair 544/590 (Figure 23).

      Figure 23. Select the right wavelength pair

    8. Click on ‘Shake1’ (Figure 24).

      Figure 24. Open the ‘Shake1’ window

    9. Set a time of 30 sec and a speed that is no more than 900 rpm (Figure 25).

      Figure 25. Settings for shaking the samples. Not too fast!

    10. Click on ‘General’ (Figure 26).

      Figure 26. Open the ‘General’ window

    11. Click on ‘area definition’ tab and select the wells to read by dragging the mouse over them (Figure 27).

      Figure 27. Area setting

      To remove wells, make sure the red well icon is click. To add wells, make sure the green well icon is clicked (Figure 28).

      Figure 28. Area setting controls

    12. Click on ‘Layout’ (Figure 29). Clear any layout on the screen by dragging the mouse over the wells then click on ‘Clear’ (Figure 29). Select ‘BLANK’ in ‘Type’ (Figure 29) then select the wells containing your untreated samples. Click on ‘Apply’ (Figure 29).
    13. Change ‘Type’ to ‘SAMPLE’ and select all the wells containing treated cells. Click on ‘Apply’ (Figure 29).

      Figure 29. Set the layout

    14. Click on the START button (Figure 30).

      Figure 30. Read your samples

Data analysis

For Procedure C (FACS analysis) and Procedure E (Fluoroskan analysis), the calculations are straightforward which involve taking the values obtained at the end of each procedure and determine the average and standard deviation. Using a reference sample and express the others as a percentage is a good idea especially if there is a need to compare different experiments (Figure 31).

Figure 31. Plotting the FACS results. Sample ‘Treated 1’ was used as the reference sample and adjusted to 100%, and sample ‘Treated 2’ was expressed relative to this reference. This graph shows that sample ‘Treated 2’ takes up 20% more drug than the reference sample.
Note: The blank sample in Figure 31 does not need to be absolutely zero, but its value must be negligible. Adjust the voltage of the DNR detector so that > 95% of the Blank cells are on the left of the line (Figure 9). It is recommended to subtract the values of the blank from all other values (e.g., zero time point).


  1. Phosphate buffer saline (PBS) pH 7.4 (1 L)
    8 g NaCl
    0.2 g mM KCl
    1.44 g mM Na2HPO4
    0.24 g mM KH2PO4
  2. Uptake buffer pH 7.4 (100 ml)
    12.5 ml 1 M NaCl
    2.0 ml 0.5 M HEPES pH 7.4
    0.6 ml 1 M glucose
    0.5 ml 1 M KCl
    0.6 ml 0.2 M KH2PO4
    0.1 1 M CaCl2
    0.6 ml 0.2 M MgSO4


This work was funded by the research grant MOP-93573 to D.R. from the Canadian Institute of Health Research. We thank Dr. Mikhail Sergeev for providing technical guidance with the microscopes and Martine Dupuis for technical guidance with the FACS. A brief version of this protocol was previously described (Andreev et al., 2016).


  1. Andreev, E., Brosseau, N., Carmona, E., Mes-Masson, A. M. and Ramotar, D. (2016). The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin. Sci Rep 6: 20508.
  2. Aouida, M., Poulin, R. and Ramotar, D. (2010). The human carnitine transporter SLC22A16 mediates high affinity uptake of the anticancer polyamine analogue bleomycin-A5. J Biol Chem 285(9): 6275-6284.
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  5. Ciarimboli, G. and Schlatter, E. (2005). Regulation of organic cation transport. Pflugers Arch 449(5): 423-441.
  6. Deng, S., Yan, T., Jendrny, C., Nemecek, A., Vincetic, M., Godtel-Armbrust, U. and Wojnowski, L. (2014). Dexrazoxane may prevent doxorubicin-induced DNA damage via depleting both topoisomerase II isoforms. BMC Cancer 14: 842.
  7. Filippo, C. A., Ardon, O. and Longo, N. (2011). Glycosylation of the OCTN2 carnitine transporter: study of natural mutations identified in patients with primary carnitine deficiency. Biochim Biophys Acta 1812(3): 312-320.
  8. Pelis, R. M., Suhre, W. M. and Wright, S. H. (2006). Functional influence of N-glycosylation in OCT2-mediated tetraethylammonium transport. Am J Physiol Renal Physiol 290(5): F1118-1126.
  9. Riganti, C., Gazzano, E., Gulino, G. R., Volante, M., Ghigo, D. and Kopecka, J. (2015). Two repeated low doses of doxorubicin are more effective than a single high dose against tumors overexpressing P-glycoprotein. Cancer Lett 360(2): 219-226.
  10. Sprowl, J. A., Ong, S. S., Gibson, A. A., Hu, S., Du, G., Lin, W., Li, L., Bharill, S., Ness, R. A., Stecula, A., Offer, S. M., Diasio, R. B., Nies, A. T., Schwab, M., Cavaletti, G., Schlatter, E., Ciarimboli, G., Schellens, J. H., Isacoff, E. Y., Sali, A., Chen, T., Baker, S. D., Sparreboom, A. and Pabla, N. (2016). A phosphotyrosine switch regulates organic cation transporters. Nat Commun 7: 10880.
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  13. Zhou, F., Xu, W., Hong, M., Pan, Z., Sinko, P. J., Ma, J. and You, G. (2005). The role of N-linked glycosylation in protein folding, membrane targeting, and substrate binding of human organic anion transporter hOAT4. Mol Pharmacol 67(3): 868-876.


蒽环类药物,如多柔比星和柔红霉素,是DNA损伤剂,其可自发荧光并且可以容易地在细胞中检测到。 在这里,我们开发了合适的测定法来定量和定位哺乳动物细胞中的柔红霉素。 可以利用这些测定来鉴定参与蒽环类药物吸收的成分。
【背景】蒽环类药物,如多柔比星和柔红霉素,通过损伤DNA起作用,用于治疗各种类型的癌症,包括急性骨髓性白血病。当癌症患者被系统地给予蒽环类抗生素时,有几个因素限制了到达肿瘤部位的药物的量(Chauncey,2001; Deng等,2014; Riganti等,2015)。在肿瘤中,对癌细胞的药物活性受到药物吸收不良,药物流出过量以及细胞靶标变化的限制。几十年来,这些DNA损伤剂如何进入癌细胞还不清楚(Aouida等,2010; Cesar-Razquin等,2015; Zhang等,2015)。为了解决这个问题,我们开发了三种可靠的体外测定法来监测柔红霉素在细胞中的积累。这些测定中的两个是定量的,需要获得荧光活化细胞分选(FACS)口径和Fluoroskan仪器,第三个是使用落射荧光显微镜进行半定量。使用这些测定法,我们确定柔红霉素以时间和浓度依赖的方式进入细胞,并且每种细胞类型显示不同的摄取速率,这表明活性过程参与蒽环类药物的摄入(Andreev等人, 2016)。我们应用了这些检测方法,发现有机阳离子转运蛋白1(OCT1)是促进柔红霉素进入细胞的关键蛋白质(Andreev et al。,2016)。调节OCT1的水平,导致对柔红霉素摄取和敏感性改变的细胞(Andreev et al。,2016)。这些测定提供了另外的转运蛋白存在以允许将柔红霉素摄入细胞的暗示。我们认为这些摄取测定法可以进一步利用来鉴定其他因素,如激酶(Tanaka et al。,2004; Ciarimboli and Schlatter,2005; Zhou et al。,2005; Pelis et al。,2006; Filippo et al。 2011; Sprowl等人,2016),其影响柔红霉素摄取的速率。在这个协议中,我们描述了三个分析来监测蒽环类药物摄入细胞。

关键字:流入转运蛋白, 蒽环类药物, 自体荧光药, FACS分析, 荧光显微镜检查, Fluoroskan读数仪


  1. 用于粘附细胞的哺乳动物细胞培养容器(100和60mm培养皿和T-75烧瓶)
    1. 但是,适用于您的细胞系的任何其他培养皿或烧瓶都可能会起作用。
    2. 请注意,您可以在贴壁细胞的容器中生长悬浮细胞。
  2. Eppendorf管
  3. 将适应流式细胞仪的FACS管:
    Falcon 5毫升聚苯乙烯圆底管(Corning,Falcon ®,目录号:352058)
  4. 显微镜幻灯片(UltiDent Scientific,目录号:170-7107A)
  5. 盖玻璃(UltiDent Scientific,目录号:170-C1818)
  6. 黑色透明平底96孔板(Thermo Fisher Scientific,Thermo ScientificTM,目录号:165305)
  7. 哺乳动物贴壁细胞,例如HeLa,HEK293和TOV112D
  8. 需要完整的培养基
    1. DMEM(WISENT,目录号:319-005-EL)
    2. 1x RPMI 1640(WISENT,目录号:350-000-CL)
    3. 卵巢表面上皮(OSE)(WISENT,目录号:316-030-CL)
    1. 胎牛血清(FBS)(WISENT,目录号:095150)
    2. 青霉素/链霉素(WISENT,目录号:450-201-EL)
    3. 硫酸庆大霉素50 mg / ml溶液(WISENT,目录号:450-135-XL)以1,000倍提供,必须在培养基中稀释1:1,000;
    4. 在OSE中使用0.5μg/ ml的两性霉素B(Sigma-Aldrich,目录号:A4888)
      注意:我们通常准备250μg/ ml的储备液,并加入1ml至500ml的OSE。
  9. 胰蛋白酶(PBS中为0.05%或0.25%)(Thermo Fisher Scientific,Gibco TM,目录号:25200072)
  10. 柔红霉素
    1. 药房(Maisonneuve-Rosemont Hospital)提供,但可以从Sigma(Sigma-Aldrich,目录号:30450)购买。在无菌水中以5mg / ml制备,最终摩尔浓度为8.87mM。/ / em>
    2. 柔红霉素可以被多柔比星代替(Sigma-Aldrich,目录号:D1515)。
  11. 4%多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)在PBS中的溶液
  12. 带DAPI的安装介质(Vectashield,Vector Laboratories,目录号:H-1200)
    注意:该组分可以被含有1至10μg/ ml Hoechst 33342(Thermo Fisher Scientific,Invitrogen)的50%甘油(Bio Basic,目录号:GB0232)替代, / em> ,目录号:H1399)。
  13. 指甲油
  14. 磷酸盐缓冲盐水(PBS)(见食谱)
    磷酸二氢钠(Na 2 HPO 4)(Bio Basic,目录号:S0404)
    磷酸二氢钾(KH 2 PO 4)(Bio Basic,目录号:PB0445)
  15. 摄取缓冲液(请参阅食谱)
    HEPES pH 7.4(Bio Basic,目录号:HB0265)
    磷酸二氢钾(KH 2 PO 4)(Bio Basic,目录号:PB0445)
    氯化钙(CaCl 2)(Fisher Scientific,目录号:C79-500)
    硫酸镁(MgSO 4)(Bio Basic,目录号:MN1988)


  1. 移液器(任何移液器都可以)
  2. 孵化器(任何孵化器如果保持5%CO 2 和湿气)就可以使用
  3. pH计(任何pH计将工作)
  4. Eppendorf离心机(任何可适应1.5 ml微量离心管的离心机)
  5. 血细胞计数器(任何血细胞计数器都可以)
  6. 流式细胞仪(FACS)
    注意:该协议正在使用Becton-Dickson的FACS Calibur(BD,型号:FACS Calibur TM )。
  7. 荧光显微镜(Olympus,型号:BX53)
  8. Fluoroskan Ascent(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Fluoroskan Ascent TM )


  1. ImageJ
    注意:我们在美国国立卫生研究院网站上使用了一个; https://imagej.nih.gov/ij/
  2. CellQuest Pro版本4.0.1© 1994-2001 BDB或5.2.1© 1994-2005 BD


  1. 细胞的制备
    1. 在37℃,5%CO 2,95%空气中培养推荐的培养基中感兴趣的细胞。
    2. 在实验前一天,从细胞中取出培养基并用PBS洗涤(参见食谱)。加入适量体积的胰蛋白酶(100毫升培养皿或T-75烧瓶1-2毫升)。在37℃下孵育2至15分钟。
    3. 向细胞添加完整的培养基以抑制胰蛋白酶。
    4. 将细胞移入60毫升培养皿中,总共约5至6毫升培养基。
      1. 所需板数取决于您要执行的实验种类。您总是需要至少一个Petri平板进行阴性对照,但最好将所有样品一式三份。每个时间点或三个板块需要三块板,以测试每种柔红霉素浓度。
      2. 你必须种细胞,所以在实验当天的汇合率低于50%,因为更高的汇合会人为地减少柔红霉素摄入。
    5. 在37℃,5%CO 2,95%空气中孵育细胞。

  2. 用柔红霉素治疗细胞
    1. 从细胞中取出培养基。
    2. 向细胞中加入5ml UB。
    3. 在37℃,5%CO 2,95%空气中孵育至少5分钟。
    4. 通过将8.87mM的库存浓度稀释35倍,在UB中准备浓度为255μM的柔红霉素稀释度。
    5. 向5ml的UB中加入100μl的25μM柔红霉素,使终浓度为5μM。
      1. 为了测试不同浓度的柔红霉素,请记住所需的稀释的柔红霉素体积,以调整添加到细胞中的UB的体积。尝试各种浓度可能是一件好事,以确定您的实验的最佳浓度。参考浓度应为5μM。
      2. 请记住,您将需要一个未处理的细胞样本进行所有操作。
    6. 将细胞在37℃,5%CO 2,95%空气中孵育所需时间。
      注意:在几分钟后可以检测到柔红霉素摄取,但较长的暴露时间可能会增加样品之间的差异。因此,为了确定实验的最佳时间点,可能需要时间依赖的曲线。 30到60分钟之间的曝光时间将是最佳起点。
    7. 一旦在37℃下孵育完成,根据细胞类型,它们可能开始从板上脱离。如果是这种情况,通过上下移液来收集细胞,以将所有细胞分离并放入Eppendorf管中。使用任何标准的Eppendorf离心机将细胞以5,000×g /孔分散1分钟,并除去上清液。用1ml冷PBS洗涤细胞。
    8. 向细胞沉淀中加入100μl的4%PFA,并在室温下孵育10分钟或在4℃下孵育60分钟。
    9. 离心5000 x g 1分钟,取出上清液。
    10. 加入100μlPBS。

  3. 处理细胞的FACS分析(需要大约100,000个细胞进行分析)
    1. 对每个样品取一个FACS管,并在每个管中放入250-500μlPBS。
    2. 将方法B中制备的细胞加入FACS管中的PBS。
    3. 打开FACS和计算机,并确保阀门关闭,以便FACS Flow罐受到压力。
      这些坦克在机器的“抽屉”中。有两个坦克,正确的是FACS Flow,左边是一个垃圾。阀门位于油箱之间。
    4. 打开FACS的软件(这里我们使用的是CellQuest Pro)。
    5. 转到“获取”标签,然后点击“连接到细胞仪”(图1)。


    6. 输入每个所需检测器的名称。使用P4(FL2 585/42)进行柔红霉素检测(图2)


    7. 选择要保存数据的文件夹,方法是单击第一个“更改”(图3)。


    8. 绘制点图或密度图,并更改“绘图类型”以获取分析(图4)。

      图4.绘制创作。 确保更改绘图类型。

    9. 点击“编辑”选项卡和“复制”创建另一个图形。在第二个图表上,将x轴名称更改为柔红霉素。
      注意:这就像做“复制/粘贴”,但是它不能在CellQuest Pro中复制图形,这就是为什么必须使用“重复”功能的原因。图形也可以逐个创建,但这需要每次更改“绘图类型”。
    10. 在“细胞计数器”选项卡中,单击“检测器/安培”。将荧光灯更改为“日志”模式(图5)。


    11. 点击象限工具,并在柔红霉素图中创建象限,将垂直线放置在10左右,水平线绕图形中间(图6)。


    12. 点击“统计”标签和“象限统计”(图7)。


    13. 从机器上取下任何管,然后按'Prime'。一旦按钮的指示灯熄灭,再按一次。
    14. 将一个阴性对照管放在机器上,按'Run'。
    15. 确保选中“设置”复选框,然后单击“获取”(图8)。


    16. 更改检测器的电压,使几乎所有的单元格都位于左下象限中。 (图9)
      1. 使用SSC作为y轴,放置象限的水平线,以排除具有很高死亡细胞机率的非常高的SSC值的细胞。因此,上方的象限将排除死细胞。
      2. 从分析中排除死细胞的另一个选择是在显示单元格(P1和P2)的FSC和SSC的图形上创建一个门。死细胞将具有非常低的FSC和高SSC。为此,在FSC-SSC图上,使用门工具选择活细胞群体。在柔红霉素图的菜单中,转到“门”,选择“R1”或相应的门。


    17. 点击“设置”复选框,在样本文本框中删除检查并写入第一个样本的名称(图10)。


    18. 将要分析的样品放在机器上,然后单击“Acquire”(图11)。


    19. 更换您的管并重复所有样品的采集。
    20. 一旦您分析了所有的样品,您可以通过点击图表将它们显示在图形中,然后在“绘图”选项卡中,您可以更改显示的数据(图12)。


    21. 要分析数据,将右下象限中的平均柔红霉素荧光乘以象限统计窗口中显示的象限中的细胞百分比(图13)。


      注意:如果您使用用荧光标记物转染的细胞系,例如,研究用GFP标记的特定蛋白质的过度表达的影响,则创建另一个图形并改变该图的x轴(FL1 530 / 30)。使用门控工具创建门,仅使用未转染细胞样本的GFP阳性细胞作为阴性对照来设置阈值。接下来,在显示柔红霉素荧光的图表的菜单中,转到Gate并选择R1。这样,显示柔红霉素的图只显示GFP阳性细胞。如果您进行瞬时转染,这可能非常有用。但是,您可能需要增加在柔红霉素图中获得足够GFP阳性细胞的事件数。为此,请访问“获取”选项卡,然后单击获取和存储。在那里你可以设置偶数。在FL2检测器上可能会出现GFP泄漏,因此使用P5(FL3)可能是一个选项。您还可以使用细胞计数器选项卡中的补偿菜单来降低GFP对FL2的影响,以及柔红霉素对F1的影响。

  4. 荧光显微镜检查
    1. 在显微镜载玻片上放置3μl带有DAPI的固定介质。
      注意:使用DAPI的固定介质可以用含有1〜10μg/ ml Hoechst 33342的50%甘油溶液代替。
    2. 从程序B中加入2μl细胞悬液
    3. 在样品上盖上盖子并用指甲油固定。
    4. 使用DAPI过滤器,用荧光显微镜观察细胞,使细胞核和红色滤光片(德克萨斯红)可视化为柔红霉素60倍或100倍。
      1. DAPI过滤器也用于染色Hoechst 33342。
      2. 为了可视化柔红霉素,您必须激发从480 nm到560 nm的波长。通过FITC或Texas RED滤波器的激励应该起作用。柔红霉素的发射可以从575 nm到680 nm(德克萨斯州红/麦克滤光片)检测。使用Texas RED滤波器组将是最好的,否则可能需要不同的发射和激发滤波器。
    5. 使用对照样品,设置光强度和曝光时间,使柔红霉素清晰可见但不饱和(图14)。


    6. 要调整光线强度,请使用与显微镜相连的灯具
    7. 曝光时间的调整在“曝光窗口”中的计算机上进行(图15) 要以定量方式使用此方法,您不得更改这些daunorubicin可视化参数。


    8. 调整这些参数(光强度和曝光时间)也适用于核 这些设置可以从一个样品更改为另一个样品,除非您想要研究染色质本身。 DAPI或Hoechst对染色质的染色可能会被大量DNR污染到DNA上。
    9. 如果使用GFP或其他颜色,请设置此颜色的光强度和曝光时间。
    10. 拍摄所有样品的所有颜色的照片。为此,您必须单击“捕捉”(图16)。


    11. 您可以通过点击图片上方的标签,在您拍摄的照片之间导航(图17)。
      1. “实时”选项卡可让您看到显微镜上的内容。要显示它,如果不是,请点击实时按钮。
      2. 重要提示:显微镜具有“模式”,因此可以通过眼睛,相机或两者显示。拍照时,请确认“模式”是使用相机。在奥林巴斯B53上,可以通过拉或拉金属棒在眼睛右侧。
      3. 一些显微镜如ZEISS Z2使用专门的软件,也可以用来编辑图片以创建具有颜色和合并图片的TIFF文件。


    12. 保存所有可以用ImageJ处理的图像。
    13. 打开ImageJ软件并点击文件>打开图像。打开单个样品的所有颜色(图18)。


    14. 点击图片,然后转到“图像”>渠道工具(图19)


    15. 在频道窗口中,点击“更多”,然后点击所选图片所需的颜色。将颜色应用于所有图片(图20)。


    16. 点击“合并通道”,并为每种颜色选择所需的图片。点击“确定”(图21)。
    17. 您现在可以按照所需的文件类型“保存为”所有图片:TIFF,JPEG,等。


  5. 氟碳分析
    1. 将细胞悬浮液从步骤B 1:10稀释至1:100,具体取决于细胞密度。
    2. 将10μl稀释液放在血细胞计数器上,并计数25个中央正方形中的细胞数,并将数乘以20,000。
    3. 然后乘以稀释因子获得的数目,以获得细胞悬浮液中每毫升的细胞数。
    4. 在黑色透明的平底96孔板中,放入6,000至20,000个细胞,并将每个孔中的体积用PBS填充至100μl。每个井必须具有相同数量的细胞。
    5. 将盘子放在Fluoroskan读板器上。
    6. 点击“Measure1”(图22)。


    7. 选择波长对544/590(图23)


    8. 点击“Shake1”(图24)


    9. 设置30秒的时间和不超过900 rpm的速度(图25)。

      图25.摇动样品的设置。 不要太快!

    10. 点击“常规”(图26)。


    11. 点击“区域定义”选项卡,然后通过将鼠标拖到其上来选择要读取的孔(图27)




    12. 点击“布局”(图29)。通过将鼠标拖动到井上,清除屏幕上的任何布局,然后单击“清除”(图29)。在“Type”中选择“BLANK”(图29),然后选择包含未处理样品的孔。点击“应用”(图29)。
    13. 将“类型”更改为“样品”,并选择所有含有处理过的细胞的孔。点击“应用”(图29)。


    14. 点击开始按钮(图30)。




注意:图31中的空白样本不需要绝对为零,但其值必须可忽略。调整DNR检测器的电压,使> 95%的空白单元格在行左边(图9)。建议从所有其他值(例如,零时间点)中减去空白值。


  1. 磷酸盐缓冲盐水(PBS)pH 7.4(1L)
    0.2 g mM KCl
    1.44g mM Na 2 HPO 4
    0.24g mM KH 2 PO 4
  2. 吸收缓冲液pH 7.4(100ml)
    12.5 ml 1M NaCl 2.0ml 0.5M HEPES pH 7.4
    0.6 ml 1 M葡萄糖
    0.5 ml 1M KCl 0.6ml 0.2M KH 2 PO 4
    0.1 1M CaCl 2
    0.6ml 0.2M MgSO 4


这项工作由研究资助MOP-93573资助给D.R.来自加拿大卫生研究所。我们感谢Mikhail Sergeev博士向显微镜和Martine Dupuis提供与FACS技术指导的技术指导。以前描述了该协议的简要版本(Andreev等人,2016)。


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引用:Brosseau, N., Andreev, E. and Ramotar, D. (2017). Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells. Bio-protocol 7(18): e2555. DOI: 10.21769/BioProtoc.2555.