2018.12.31: Coating and seeding
- Prepare 15 plates of 100 mm of diameter coated with laminin for 1h (2’4 microg/ml diluted in Borate Buffer) –> in this case, the laminin is at 0’5 mg/ml, so we put 4’4 microl laminin per ml of Borate Buffer.
- Wash with PBS 1x (3 times)
- Seed aprox 15000 cells/cm2 –> every plate has 58 cm2 –> 800000 cells/plate 100 mm (in proliferation medium)
- Incubate at 37ºC
2019.01.02: proliferation medium exchange
Change the medium of the cells –> new proliferation medium
2019.01.04: treatment and medium exchange (differentiation)
Change the medium of the cells –> Add retinoic acid to the differentiation medium (1 microl RA in 1 ml differentiation medium)
*Change the half of the differentiation medium (+ retinoic acid) every 2 or 3 days –> 07.01.19 / 09.01.19 / 11.01.19
2019.01.14: Immunoprecipitation with Magnetic beads
Pierce Magnetic IP Kit
- Incubate the culture with the serum of the patient to be analyzed 1h at 37ºC (in the incubator). Dilution is done in the culture medium itself (on the plate)
- For IgG: 1/100 -> 30 μl serum in 3 ml medium
- Wash 1 time with PBS1x
- Add 800 μl of IP Lysis / Wash buffer + protease inhibitors to each plate. Leave them in strong agitation during 5 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 100 μl of Lane Marker Sample Buffer (diluted 5x in distilled water) to the tube and heat to 96 – 100 ° C for 10 minutes
- The amount of Lane Marker Sample Buffer can be changed depending on the objective of the IP.
- 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)
