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2021-02-01T19:53:45Z

Created page with "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 sy..."

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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:''' 

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

= Protocol - Receptor Activation =

#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. 
#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.
<pre>Note: Temperatures below 15C will inhibit phosphatase activity more than kinase activity, thereby amplifying the effects of receptor cross-linking.
</pre> <pre>Note: If a cytoskeletal pathway is suspected, do not cool cells below 10C, as this will depolymerize microtubules.
</pre>
#Add primary antibody at a saturating concentration (generally 1:100-1:500), 20 min, 10C in HPMI-buffered serum-free media.
#Wash 3X with 10C PBS.
#Add secondary antibody at saturation concentration (generall 1:100-1:500), 20min, 10C in HPMI-buffered serum-free media..
#Wash 3X with PBS or media.
#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: 

#Wash cells with PBS to remove any residual media. 
#Fix cells for 20 min, on-ice, with 4% paraformaldehyde in PBS. 
#Permeabilze and stain with 4G10 antibody as per the [[Immunostaining|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: 

#Aspirate cold media/PBS completely from the well. 
#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. 
#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. 
#After the incubation period, rapidly cool the plate and terminate internalization through 2-3 large-volume washes (75% or well capacity) with 10C PBS. 
#Remove any surface-bound antibody through the addition of pH 2.0 PBS, 10C for 2 min. 
#Was 2X with 10C PBS, then fix for 20 min at 10C with 4% paraformaldehyde in PBS.
#Co-stain with cholera toxin or wheat-germ agglutinin, if required.

= Known Working Conditions =

{| cellspacing="1" cellpadding="1" border="1" width="100%"
|-
| '''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
|}

Receptor Cross-linking and Activation - Revision history

Admin: Created page with "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 sy..."