Receptor Cross-linking and Activation

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Ligand-independent activation of most receptors can be achieved through antibody-mediated cross-linking.  However, care must be taken, especially on cels of the immune system, to activate receptors in a way which avoids activating the Fc receptors which normally bind to antibodies.  This can be achieved by using Fab's derived from the primary antibody and F(ab')2 seocndaries to cross-link, or less preferably, cells can be pre-treated with Fc blocking antibodies.

Notes:

  1. Prior to the experiment cells should be cultured as per usual, aiming for 60%-80% confluency.

Protocol - Receptor Activation

  1. Serum starve cells for 3 hours prior to experiment in order to reduce basal levels of endocytosis. Treat with any inhibitors at the end of this process.
  2. Replace media with HPMI-buffered serum-free at 4C or 10C.  Place on-ice (4C) or in 10C incubator and let equliberate for 5 min.
Note: Temperatures below 15C will inhibit phosphatase activity more than kinase activity, thereby amplifying the effects of receptor cross-linking. 

Note: If a cytoskeletal pathway is suspected, do not cool cells below 10C, as this will depolymerize microtubules.
  1. Add primary antibody at a saturating concentration (generally 1:100-1:500), 20 min, 10C in HPMI-buffered serum-free media.
  2. Wash 3X with 10C PBS.
  3. Add secondary antibody at saturation concentration (generall 1:100-1:500), 20min, 10C in HPMI-buffered serum-free media..
  4. Wash 3X with PBS or media.
  5. Move onto "Internalization" or "Phosphotyrosine Immunostaining" as required.


Phosphotyrosine Staining

In many cases we wish to to image receptor activation following cross-linking. This works best if the 'receptor activation' portion of this protocol is performed at 4C. Once step 6 (above) is compelte:

  1. Wash cells with PBS to remove any residual media.
  2. Fix cells for 20 min, on-ice, with 4% paraformaldehyde in PBS.
  3. Permeabilze and stain with 4G10 antibody as per the standard immunostaining protocol for permeablized cells.


Internalization Assay

To quantify receptor internalization the receptor activation protocol must be performed at 10C to avoid cold-temperature effects on the cytoskeleton.  Prepare and pre-cool pH 2.0 PBS (30ml minimal volume) prior to begining experiment. Once step 6 (above) is complete:

  1. Aspirate cold media/PBS completely from the well.
  2. Gently wash the well 2X with a large (75% of well volume) amount of 37C HPMI-buffered serum free media. Do this gently as the goal is to rapidly warm the sample, and is not to remove antibody or cells from the sample.
  3. After second wash place normal amount of 37C serum-free media into well and transfer to 37C incubator for desired internalization time (usually 20 min). Use HPMI-buffered media if incubating in air, bicarbonate-buffered media if placing in CO2-incubator.
  4. After the incubation period, rapidly cool the plate and terminate internalization through 2-3 large-volume washes (75% or well capacity) with 10C PBS.
  5. Remove any surface-bound antibody through the addition of pH 2.0 PBS, 10C for 2 min.
  6. Was 2X with 10C PBS, then fix for 20 min at 10C with 4% paraformaldehyde in PBS.
  7. Co-stain with cholera toxin or wheat-germ agglutinin, if required.


Known Working Conditions

Antibody Primary Concentration Secondary Antibody Secondary Concentration Notes
CD36 IgA (clone 63) 1:100, serum-free media anti-mouse F(ab')2, jackson immuno 1:100, serum-free media IgA antibodies do not activate Fc receptors


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