Protocol to measure the recycling of MHCI to the PM
The protocol is based on accumulating MHCI intracellularly in the presence of Latrunculin A, which is a reversible inhibitor of MHCI recycling. Upon removal of the drugs MHCI recycles back to the PM 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).
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:
- Binding mixture (50µl/coverslip): 50mg/ml of W6/32 antibody (mouse anti-human HLA-A,B,C monoclonal antibody (w6/32) BioLegend (San Diego, CA)) in complete DMEM-HEPES (DMEM with 10%FCS; For 1 ml medium add 25 µl 1M Hepes PH 7.4)
- Uptake mixture (50µl/coverslip): complete DMEM containing 1µM Latrunculin A (stock solution is 1mM LatA in DMSO, store at -20ºC)
- Recycling mixture: complete DMEM
- 3.7% paraformaldehyde in PBS
- 3% BSA in PBS (=blocking solution)
- 10% saponin in water
- Stripping buffer (14.6 g NaCl, 2.5 ml Acetic Acid, 500 ml distilled water)
- Prepare 2 large Petri dishes (one for the binding and one for the uptake), put the parafilm, spot 50 µl of the appropriate mixture
Each sample will need 4 coverslips – 2 time points, each 2 coverslips (surface and total MHCI pools). Since the stripping step is very short, I handle no more than 4-6 coverslips at any time point.
- For the binding, place the coverslips upside-down on the drops with the binding mixture for 30 minutes on ice.
(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 complete DMEM/well into 24-well plate for the recycling and transfer the plate to 37ºC, 5%CO2 incubator).
- Wash the coverslips in ice cold PBS twice
- Put the coveslips upside-down on the drops with the uptake mixture and incubate for 30 minutes at 37ºC, 5%CO2.
- Wash the coverslips in PBS at R.T. twice
- Wash the coverslips for 30-40 sec in stripping buffer
- Wash the coverslips twice in PBS at R.T.
- Wash the coverslips twice in DMEM-HEPES
- Fix a set of coverslips in 3.7% formaldehyde for 15 min R.T. (=time 0min=uptake), proceed to step 13.
- For the recycling put the second set of coveslips in the recycling mixture (complete DMEM) for 30 minutes at 37ºC, 5%CO2.
- At the end of the incubation wash the coverslips twice in PBS
- With one set of duplicate coverslips proceed to step 12. Wash second set of duplicate coverslips for 30-40 sec in stripping buffer, followed by 2 washes in PBS (In some cases, depending on the way cells were treated (transfection, siRNA), cells tend to detach from coverslips upon second treatment with the stripping buffer, in that case I proceeded straight to fixing step).
- Fix the coverslips in 3.7% formaldehyde for 15 min R.T. (=time 30min=recycling).
- wash the coverslips 3 times in PBS
- Block for at least 30 minutes with 3%BSA in PBS
- Incubate for 1h with Alexa 568/488 goat anti-mouse secondary Ab (1:500) in blocking solution in the presence (total pool) or absence (surface pool) of 0.2% Saponin
- Wash 3-5 times in PBS (at least 5 minutes per wash)
- Mount the coverslips on a glass slide (I use vectashield mounting media) and seal with cytoseal60.
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 MHCI and other 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). in the original paper they expressed recycling as follows:
- Each time Total values= surface(no saponin) + internal MHCI(+saponin)
- Express surface MHCI as a percentage of total MHCI (=surface MHCI/(surfaceMHCI+internal MHCI) x 100%)
- Amount of recycled MHCI is % at 30min - % at 0min
- 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).