Cultures: An In Vitro Tool to Identify Demyelinating and Axopathic Autoantibodies



Fig. 1
Representative immunofluorescence images of myelinating cultures (28 DIV). (a) Two colour immunofluorescence microscopy reveals the presence of a dense bed of neurites (Red; neurofilament-specific antibody SMI31) and myelinating oligodendrocytes (Green; myelin oligodendrocyte glycoprotein-specific antibody Z2). Note only a relatively small proportion of neurites are myelinated and myelin sheaths are not present along their full length. (b) Cells of the oligodendrocyte lineage can also be identified using antibodies specific for the transcription factor Olig2 (Red). (c) Representative immunofluorescence image obtained using the PLP/DM20-specific antibody AA3 (Green)




 


2.

Confirm cultures are well myelinated: axonal density should be at least 70 %, myelination >8 % (see Notes 3 and 9 ).

 

3.

Add test sample in the presence and absence of rabbit serum as a source of complement (see Notes 2 and 4 8 ).

 

4.

Incubate for 16 h at 37 °C in 7.5 % CO2.

 

5.

For fixation, staining, and analysis protocols see below.

 



Monitoring Changes Induced by Soluble Factors Over Time

1.

To assess effects on myelination cultures are treated with the factor of interest diluted in culture medium from 18 to 28 DIV (see Note 3 ).

 

2.

To investigate effects on neurite outgrowth or OPC numbers the experiment should be initiated at time points between 6 and 12 DIV.

 

3.

Feed cultures three times a week with appropriate medium in the presence or absence of the factor of interest.

 

4.

Harvest coverslips at different time points. For fixation, staining and analysis protocols see below.

 

5.

To assess effects on cells of the oligodendrocyte lineage use appropriate combinations of stage-specific markers to identify early and late progenitors (Olig2, A2B5, PDGFRα, NG2, O4), pre-myelinating oligodendrocytes (PLP/DM20), and terminally differentiated/myelinating oligodendrocytes (MOG) (see Note 9 ).

 


Fixation and Staining

1.

Fix cells by putting coverslips directly into 4 % PFA for 20 min after which they are washed three times in PBS.

 

2.

Permeablize cells with 0.5 % Triton X100 for 10 min.

 

3.

Wash three times in PBS.

 

4.

Incubate in blocking buffer for 60 min.

 

5.

Incubate with primary antibodies diluted in blocking buffer for 45 min.

 

6.

Wash three times in PBS.

 

7.

Incubate with appropriate fluorophore-conjugated species/isotype-specific secondary antibodies diluted in blocking buffer for 15 min in the dark.

 

8.

Wash three times in PBS.

 

Jul 12, 2017 | Posted by in NEUROLOGY | Comments Off on Cultures: An In Vitro Tool to Identify Demyelinating and Axopathic Autoantibodies
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