This is the protocol to screen for Tf uptake and early endosome abundance (EEA1 staining) in 384-well plates. The protocol can be automated on the Agilent Bravo and the BioTek EL406 liquid handling devices.

This protocol was designed by Prisca Liberali.

 

Note that the concentration of Tf used and the incubation time can be different for different cell lines.

 

  1. Thaw the CellPlate. In this case, these are pre-made 384-well plates with the right amount of siRNAs aliquoted in each well. Before opening the seal, centrifuge for 1 min to spin the siRNAs at the bottom of the plate.
  2. Add the carrier mix. Mix 4.95 µl of Optimem + 0.05 µl Lipofectamine 2000 per well in an eppendorf or falcon tube. Wait 15-20 min. Add 5 µl per well to the plates. For 4 plates: 9.5 ml Optimem + 96 µl Lipofectamine 2000.
  3. Plate the cells. Plate ~1000 cells in 40 µl of complete medium +PenStrep (important to avoid contamination of your cells) per well. When doing large-scale cell-based screens, we rely on the fact that we never use any antibiotics in our tissue culture, but use PenStrep specifically and only in a large-scale screen. Like this, we do not have PenStrep-resistant bacteria in our incubators and hoods, and can use the pressure of these antibiotics to make sure no contaminations occur in such screens.
  4. After 3 days (~72h) the cells are ready for the experiment.
  5. Add Tf-Alexa488 to the cells. Add 25 µl per well of SFM (serum-free medium) + 1:300 (HeLaMz) or 1:150 (A431) of the Tf-Alexa488 stock (stock concentration= 5mg/ml) (from Molecular Probes/Invitrogen) to a final concentration of 1:1000 (5 µg/ml- HeLaMz) or 1:500 (10 µg/ml).
  6. Leave the Tf solution on the cells for 30 min at 37°C in the incubator. This time point is used to saturate the uptake and recycling pathways of Tfn, but may not be the best time-point for sensitive studies of  cell-to-cell variability, or for a specific analysis of uptake vs. recycling. Pulse-chase for different time points, using different colours of fluorescence Tf, can address such questions.
  7. Wash once with PBS at RT.
  8. Fix the cells with 4% PFA for 15 min at RT. I prefer the PFA (EM grade, 16% stock from Electron Microscopy Sciences) than the formaldehyde 37%.
  9. Permeabilize the cells with 0.1% Triton in PBS for 5 min at RT. Also Saponin 0.25% for 30 min can be used for permeabilizing.
  10. Block the cells and quench the PFA. Use a blocking solution with 1% BSA and 50 mM NH4Cl. Also serum can be used to block.
  11. Incubate the cells with 1°Ab. Dilute the EEA1 mAb (1:1000) in 20-25 µl of blocking solution per well for 1.5 h at RT. Put the plate on the shaker during the incubation.
  12. Wash 2x with PBS. Every wash should be long (~30 min per wash).
  13. Incubate the cells with 2°Ab. Dilute the mAb-Alexa546 (1:500) in 20-25 µl of blocking solution per well for 1 h at RT. Put the plate on the shaker during the incubation.
  14. Wash 2x with PBS. Every wash should be ~10 min .
  15. Stain the nuclei. Incubate the cells with DAPI (1:1000 from the stock solution of 100 µg/ml) for 5 min at RT. Put the plate on the shaker during the incubation.
  16. Stain the cells. Incubate with CellTrace (1:10000 from stock solution 1 µg/µl in DMSO) (from Molecular Probes/Invitrogen) in 100 mM Na2HCO3 for 5 min at RT. Put the plate on the shaker during the incubation. The cell staining is not very stable, if possible I prefer to stain the cells only before the image acquisition.
  17.  Wash 2x with PBS. Every wash should be ~10 min .
  18. Leave the cells in 75 µl PBS at 4°C.
  19. Image acquisition. Acquire 30 fields per well with 20x objective using the Molecular Devices ImageXpress. To have the best resolution I acquired 3-4 z planes (one every 1 µm) per image and I saved the Maximum Intensity Projection (MIP).
  20. Start Robocopy. Robocopy needs to move the images to a server during the acquisition otherwise the local computer fills up quickly.