Table 1.
Fluorescent Drug Uptake and Accumulation Assay Protocol in Multicellular Tumor Spheroids Formed from Head and Neck Cancer Tumor Cell Lines
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Rehydrate 384-well ULA plates | 50 μL of serum-free DMEM, 15 min | Rehydrate ULA-plate wells by the addition of 50 μL of serum-free DMEM to each well and incubation in a humidified incubator for 15 min. After incubation, the media are removed and 15 μL of culture medium is added to each well. |
| 2 | Harvest and centrifuge Cal33 or FaDu cells | 5 min, 500 × g | Aspirate medium, wash with PBS, trypsinize cells, add DMEM medium +10% FBS, and centrifuge |
| 3 | Viable Cal33 or FaDu cell count | Viable cell count | Re-suspend cells in culture medium and count the number of trypan blue excluding viable cells in a hemocytometer |
| 4 | Adjust Cal33 or FaDu cells to the required density and seed into 384-well ULA assay plates | Add 45 μL of cells/well and centrifuge at 100 rpm for 1 min | Seed wells at 625, 1,250, 2,500, 5,000, 10,000, or 20,000 cells/well in rehydrated 384-well ULA plates, centrifuge, and incubate overnight at 37°C, 5% CO2, and 95% humidity |
| 5 | Transfer fluorescent test compounds/DMSO to control wells | 5 μL | 0.781–25 μM final concentration in well, 0.25% DMSO |
| 6 | Incubate assay plates | 2.5–45 min | At 37°C, 5% CO2, and 95% humidity |
| 7 | Fix cells | 50 μL | 7.4% Formaldehyde containing 4 μg/mL Hoechst 33342 in Ca2+- and Mg2+-free PBS pre-warmed to 37°C |
| 8 | Incubate assay plates | 30 min | Ambient temperature |
| 9 | Aspirate fixative and wash 2 × with PBS | 50 μL | Aspirate fixative and wash twice with 50 μL Ca2+- and Mg2+-free PBS, 50 μL PBS in well |
| 10 | Seal plates | 1 × | Seal with adhesive aluminum or clear plate seals |
| 11 | Acquire images | 4 × , 0.2NA objective | Transmitted light and fluorescent images of the DAPI (Ch1), FITC (Ch2), and TRITC (Ch3) channels were sequentially acquired on the ImageXpress® Micro platform by using a 300 W Xenon lamp broad spectrum white light source, ZPS filter sets, and a 1.4 megapixel 2/3" chip Cooled CCD Camera. |
| 12 | Image analysis assay readout | Mean integrated fluorescent intensity values | Images were analyzed by using the Multi-Wavelength Cell Scoring image analysis module and by using the mean integrated fluorescent intensity values to quantify uptake and accumulation. |
Step Notes
1. The treated surface of the 384-well ULA plates was rehydrated by incubation with 50 μL of serum-free medium for 15 min—this step reduced the number of failed wells. After incubation, removing the serum-free medium and replacing with 15 μL culture medium prevents the ULA surface from drying out while cell suspensions are prepared.
2. Cal33 or FaDu cells were maintained in DMEM medium with 2 mM l-glutamine supplemented with 10% FBS, and 100 U/mL penicillin and streptomycin in a humidified incubator at 37°C, 5% CO2, and 95% humidity. Cell monolayers (<70% confluent) were washed 1 × with PBS, and they were then exposed to trypsin-EDTA until they detached from the surface of the tissue culture flasks. Cells were pelleted at 500 × g for 5 min in Sorvall ST 16 Centrifuge with a TX-400 Rotor.
3. Aspirate medium, re-suspend pelleted cells in tissue culture medium+FBS, and count the number of trypan blue excluding viable cells in a hemocytometer.
4. Cal33 or FaDu head and neck cancer cell lines were seeded into 384-well ULA plates (Corning, Tewksbury, MA) at the indicated seeding densities per well and incubated for 24 h at 37°C, 5% CO2, and 95% humidity in DMEM with 2 mM l-glutamine supplemented with 10% FBS, and 100 U/mL penicillin and streptomycin.
5. 0.781–25 μM of compounds were added to wells in columns 3–22 by using a Janus MDT Mini automated liquid handler platform equipped with a 384-well transfer head.
6. Incubate treated Cal33 or FaDu cells for 2.5–45 min at 37°C, 5% CO2, and 95% humidity.
7 and 8. Fix MCTSs for 30 min at ambient temperature by adding 50 μL of 7.4% formaldehyde containing 2 μg/mL Hoechst 33342 in Ca2+- and Mg2+-free PBS pre-warmed to 37°C.
9. Aspiration of media and fixative, and PBS washes were performed by using a Janus MDT Mini automated liquid handler platform equipped with a 384-well transfer head.
10. Plates were sealed with adhesive aluminum or clear plate seals.
11. Plates were loaded into the ImageXpress Micro HCS platform (Molecular Devices, LLC, Sunnyvale, CA) for scanning.
12. Images were analyzed by using the Multi-Wavelength Cell Scoring image analysis module of MetaXpress (Molecular Devices, LLC).
Ch1, fluorescent channel 1; Ch2, fluorescent channel 2; Ch3, fluorescent channel 3; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HCS, high-content screening; MCTSs, multicellular tumor spheroids; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine; ULA plates, ultra-low attachment microtiter plates; ZPS, Zero Pixel Shift.