2019.04.25: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 : 60 mm plate = 21 cm2
- In this case –> 6 plates of 60 mm with 200.000 cells (with 26 coverslips)
2019.04.26: 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).
2019.05.03: ICC neuroblastoma neurons (day 8 of differentiation)
Samples:
- 1 – 55: IgM
- 56 – 104: IgG
Sample | Result | Sample | Result | ||
1 | 18-525 | 53 | JGV 222-24 | +++ | |
2 | 18-526 | 54 | 387 (MS) | ||
3 | 18-527 | rep. | 55 | Neg. control | |
4 | 18-528 | 56 | 2 (small fib.) | ||
5 | 18-529 | 57 | 3 (small fib.) | ||
6 | 18-530 | 58 | 8 (small fib.) | ||
7 | 18-531 | + | 59 | 13 (small fib.) | |
8 | 18-532 | 60 | 20 (small fib.) | ||
9 | 18-533 | 61 | 22 (small fib.) | ||
10 | 18-534 | 62 | 41 (small fib.) | ||
11 | 18-535 | 63 | 43 (small fib.) | ||
12 | 18-536 | 64 | 46 (small fib.) | ||
13 | 18-537 | 65 | 48 (small fib.) | ||
14 | 18-538 | 66 | 51 (small fib.) | ||
15 | 18-539 | 67 | 53 (small fib.) | ||
16 | 18-541 | 68 | 55 (small fib.) | ||
17 | 18-542 | 69 | 57 (small fib.) | ||
18 | 18-543 | 70 | 58 (small fib.) | ||
19 | 18-544 | 71 | |||
20 | 18-545 | 72 | 19-043 | ||
21 | 18-546 | 73 | 387 (MS) | ||
22 | 18-547 | 74 | JGV 09-5769 | ||
23 | 18-548 | 75 | JGV 142-2 | ||
24 | 18-549 | + | 76 | JGV 222-24 | ++ |
25 | 18-550 | 77 | (MS) 149 | ||
26 | 18-551 | + | 78 | (MS) 150 | |
27 | 18-552 | 79 | (MS) 151 | ||
28 | 18-553 | 80 | (MS) 152 | + | |
29 | 18-554 | 81 | (MS) 153 | ||
30 | 18-555 | 82 | (MS) 159 | ||
31 | 18-556 | 83 | (MS) 163 | ||
32 | 18-557 | 84 | (MS) 170 | ||
33 | 18-558 | 85 | (MS) 176 | ||
34 | 18-559 | 86 | (MS) 201 | ||
35 | 18-560 | 87 | (MS) 204 | ||
36 | 18-561 | + | 88 | (MS) 231 | |
37 | 18-562 | 89 | (MS) 240 | ||
38 | 18-563 | 90 | (MS) 241 | ||
39 | 18-564 | 91 | (MS) 251 | ||
40 | 18-565 | 92 | (MS) 254 | ||
41 | 18-566 | ? | 93 | (MS) 257 | |
42 | 18-567 | 94 | (MS) 262 | ||
43 | 18-568 | 95 | (MS) 274 | ||
44 | 18-569 | 96 | (MS) 290 | ||
45 | 18-572 | ? | 97 | (MS) 293 | |
46 | 18-573 | 98 | (MS) 295 | ||
47 | 18-574 | 99 | (MS) 298 | ||
48 | 18-575 | 100 | (MS) 318 | ||
49 | 18-577 | 101 | (MS) 325 | ||
50 | 18-578 | ++ | 102 | (MS) 326 | |
51 | JGV 09-5769 | 103 | Neg control | ||
52 | JGV 142-2 | 104 | Pos. control 15-198 | ++ |
- Fix for 15 minutes with PFA 4 %
- Wash 3 times with PBS1x
- Block for 1 hour with Goat Serum 5 %
- Patient’s sera diluted 1/100 for IgG and 1/40 for IgM (1h) –> in the box with parafilm, with the cells above
- Wash 3 times with PBS1x
- Secondary antibody: GAH488 IgG or IgM diluted 1/1000 (1 h) –> in the box with parafilm, with the cells above
- Wash 3 times with PBS1x
- Vectashield mounting medium
RESULTS: las neuronas no estaban demasiado bien (se despegan y se rompen). Repetir! (intentar mejorar el método)