2019.03.28:seed cells (in proliferation medium)
- Coating with laminin 2’5 µg/ml diluted in Borate Buffer (for 1 h at 37ºC)
- Wash 2-3 times with PBS
- Seed aprox 10.000 cels/cm2 : 150 mm plate = 150 cm2
- In this case –> 4 plates of 150 mm with 1.5 million cells
2019.03.29: differentiation
- Change all the medium of the cells (differentiation medium)
- Each time the new differentiation medium is changed, 10 μM retinoic acid (RA) is added –> stock solution -80ºC (diluted in ethanol 96 %): 0,01 M –> put 1 µl in every ml of differentiation medium (if we change the half of the medium, we have to add the double of RA)
- Once the differentiation has begun, change the half of the differentiation medium every 2 or 3 days (for 6 to 8 days).
2019.04.04: Immunoprecipitation with Magnetic beads (day 7 of differentiation)
Pierce Magnetic IP Kit
- Incubate the culture with the serum of the patient to be analyzed 1h at 37ºC (in the incubator). Dilution (1/100) is done in the culture medium itself (on the plate)
- 2 plates: 15-198
- 2 plates: negative control
- Wash 1 time with PBS1x
- Add 1 ml of IP Lysis / Wash buffer + protease inhibitors to each plate. Leave them in strong agitation during 30 min at 4ºC.
- Transfer the lysate to a 2 ml eppendorf (in each eppendorf we will have the lysate of two plates) and centrifuge 10 minutes at 13000 g
- To prepare the magnetic beads: Put 25 μl of Pierce Protein A / G Magnetic Beads in the number of eppendorfs that we will use.
- Add 175 μl of IP Lysis / Wash Buffer and vortex (gently)
- Put the tube in the magnetic support. Remove the supernatant (without removing the eppendorf from the support)
- Add 1 ml of IP Lysis / Wash Buffer and vortex (gently). Put back in the magnetic support and remove the supernatant.
- Add the antigen-antibody mixture (first part) to the eppendorf with the washed magnetic beads and incubate at room temperature for 1 hour in agitation (rotation).
- Put the eppendorfs on the magnetic support and remove the supernatant.
- Add 500 μl of IP Lysis / Wash buffer (with protease inhibitors) and mix. Put the eppendorfs on the magnetic support and remove the supernatant.
- Repeat washing
- Add 500 μl of ultra pure distilled water and mix. Put the samples on the magnetic support and remove the supernatant.
- Add 80 μl of Lane Marker Sample Buffer (diluted 5x in distilled water) to the tube and heat to 96 – 100 ° C for 10 minutes
- Put the eppendorfs in the magnetic support (the magnetic beads will separate from the antigen-antibody solution)
- With the supernatant perform the acrylamide gel electrophoresis
(save the samples at -20ºC until the electrophoresis is done)
