PROTOCOLS       

Protocol to measure uptake and recycling of Tf in HeLa cells

Protocol is slight modification of the protocol described in the reference 1.

Day 1:
Seed the cells on 12mm coverslips in 24-well plate at a density 10,000-11,000 cells/well (the goal is to have 40-50% confluency after 48 hrs).

Day 2:
Transfect the cells with DNA. I use jetPEI reagent (Polyplus-transfection, San Marcos, CA), using ½ of the suggested volumes (see manufacturer’s protocol).

Day 3: Assay
Prepare the following:

  • Serum-free DMEM containing 0.5% BSA
  • Uptake mixture (50µl/coverslip): serum-free DMEM-BSA containing 5mg/ml Alexa-568-Tf.
  • Recycling mixture: complete DMEM supplemented with non-labelled Transferrin (for 1 ml of media use 10 ml of 10mg/ml stock) and deferoxamine mesylate (for 1 ml of media add 0.7ml of 50mg/ml stock)
  • PBS
  • 3.7% paraformaldehyde in PBS
  • 3% BSA in PBS (=blocking solution), only if using protein with non-fluorescent tag
  • 0.1% TritonX-100 in PBS, only if using protein with non-fluorescent tag
  • Stripping buffer (14.6 g NaCl, 2.5 ml Acetic Acid, 500 ml distilled water)
  • Prepare 1 large Petri dishes for the uptake, put the parafilm, spot 50 µl of the Uptake mixture

Each sample will need 2 coverslips – one coverslip for the uptake and one for the recycling. Since the stripping step is very short, I handle no more than 4-6 coverslips at any time point.

  • Prior to internalization, serum-starve cells in serum-free DMEM containing 0.5% BSA for 30 minutes at 37ºC, 5%CO2. (At the same time transfer Petri dish with the drops of uptake mixture to 37ºC, 5%CO2 incubator for 30 minutes. Also put 1ml of recycling mixture/well into 24-well plate for the recycling and transfer the plate to 37ºC, 5%CO2 incubator).
  • For the uptake, place the coverslips upside-down on the drops with the uptake mixture and transfer to 37ºC, 5%CO2 incubator for 30 minutes.
  • Wash the coverslips in PBS at R.T. twice
  • Wash the coverslips for 30-40 sec in stripping buffer
  • Wash the coverslips 4x in PBS at R.T.
  • Fix a set of coverslips in 3.7% formaldehyde for 15 min R.T. (=uptake), proceed to step 10.
  • For the recycling put the second set of coveslips in the recycling mixture for 30 minutes at 37ºC, 5%CO2.
  • At the end of the incubation wash the coverslips twice in PBS
  • Fix as in step 6.
  • Wash the coverslips 4x in PBS
  • Mount the coverslips on a glass slide (I use vectashield mounting media) and seal with cytoseal60.

If transfecting the cells with protein which has non-fluorescent tag, after fixing, wash the cells 4x in PBS, permeabilize with 0.1% TritonX-100 in PBS for 15 min at R.T., wash 2x in PBS, block for 30 min at R.T with 3% BSA in PBS, stain with primary antibody diluted in 3% BSA in PBS for 1 hour at R.T., wash about 4 times (at least 5 min each time) in PBS, block as above and stain with secondary antibody diluted in 3% BSA in PBS for 1 hour at R.T., in dark, wash again about 4 times (at least 5 min each time) in PBS and mount as above.

For the quantification I take snapshot images of fields of at least 100 transfected cells per set, using 40x oil objective. Images are taken in 2 channels, one for Alexa-568-Tf and other (FITC) to see which cells are transfected. Quantification of signal was done using Metamorph software. In short, outline the transfected cells, threshold, and measure integrated intensity, which can be found in regions measurement menu. Integrated intensity is then normalized per cell area. Since absolute values tend to vary a lot from experiment to experiment, experimental values can be expressed relative to control (control value = 1).

References

  1. Weigert, R. & Donaldson, J. G. Fluorescent microscopy-based assays to study the role of Rab22a in clathrin-independent endocytosis. Methods Enzymol 403, 243-53 (2005).