Uptake Assays to Monitor Anthracyclines Entry into Mammalian 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 )
   Notes:
   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.
   
   4. 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.
      
   5. 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.
      
   6. 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
      
   7. 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, Gibco^TM, catalog number: 25200072 )
   
10. Daunorubicin
    Notes:
    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, Invitrogen^TM, 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 )
    

Equipment

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 Calibur^TM ).
   
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 Scientific^TM, model: Fluoroskan Ascent^TM )
   

Software

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
   

Procedure