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MV1p12 ECIS experiment using serum free media

May 10th, 2009

After 72 hours, MVR1p12 cells were ready for experiments using ECIS arrays.
400uL of serum free media with DMEM and 1% PCN/ST was exchanged with existing media and incubated for 2.5 hours.
ECIS arrays were connected and data recorded for 2 hours to establish baseline.
100uL of serum free media was removed from each ECIS well and replaced with 200uL of media+CSE+T10 to achieve concentrations according to previous experiments.

tung Uncategorized , , ,

Wound Assay using MV1p12, 10% CSE and serum free media

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  • May 10th, 2009

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  • MV1p12 cells were ready for wound assay with 100% confluence 72 hours after plating into 35mm plate.
    2mL Serum free media using DMEM + 1% PCN/ST was exchanged into 35mm plate.
    200uL of CSE was added to achieve 10% CSE concentration.
    Wound simulated using 200uL pipette tip and placed under video scope for 24 hours.

    tung Uncategorized , ,

    MV1p10 cells seeded 4/13/09 at 100k ready at 54 hrs

    April 15th, 2009

    Resistances as follows:

    Holder A - MV1p10 @ 100k/well

    1. 1300
    2. 1231
    3. 1299
    4. 1417
    5. 1362
    6. 1251
    7. 1343
    8. 1379

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    Holder B - MV1p10 @ 100k/well

    1. 1335
    2. 1568
    3. 1285
    4. 1421
    5. 1419
    6. 1495
    7. 1380
    8. N/A

    Will use serum-free DMEM with 1% pcn/strp for experiments with additives as follows, after complete removal of DMEM with FBS from ECIS wells:

    Additive 1 2 3 4 5 6 7 8
    T10 0 4 4 4 4 0 0 0
    CSE 0 0
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  • 100
    15 50 100
    DMEM 500 496 481 446 396 485 450 400

    tung ECIS , , ,

    MV1p9 cells ready for ECIS experiment at 104 hours

    April 3rd, 2009

    Resistances for MV1p9 cells seeded on 3/30/09 at 60k cells/well are:

    Holder A

    1. 1228
    2. 1247
    3. 1265
    4. 1206
    5. 1201
    6. 1239
    7. 1242
    8. 1246

    Holder B

    1. 1241
    2. 1150
    3. 1314
    4. 1391
    5. 1293
    6. 1310
    7. 1328
    8. 35473 -Faulty electrode

    Will plate Holder A using PBS, T10, & CSE as follows:

    Additive 1 2 3 4 5 6 7 8
    PBS 150 146 131 96 0 135 100 0
    T10 0 4 4 4 4 0 0 0
    CSE 0 0 15 50 150 15 50 150
    DMEM 100 100 100 100 100 100 szlachetne zdrowie kochanowskiego 45
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  • 100
    100

    Holder B will use 10% and 30% CSE in wells 6 & 7, omitting 3% CSE.

    tung ECIS , , , ,

    MV1p9 not ready at 48 hours post seeding

    April 1st, 2009

    The 35mm dish for the wound assay is ~50-60% confluent after 48 hours at a seed density of 100k cells.

    The resistances for MV1p9 at 60k per ECIS well are:

    Holder A:

    1. 1106
    2. 1111
    3. 995
    4. 1161
    5. 998
    6. 1047
    7. 1151
    8. 1097

    Holder B:

    1. 1057
    2. 1105
    3. 1115
    4. 1153
    5. 1134
    6. 1166
    7. 1190
    8. 34819 (Faulty electrode)

    tung ECIS , ,

    Method for cigarette smoke extract production

    April 1st, 2009
    1. 500ml vacuum bottle filled with 200ml of PBS was connected to suction apparatus with suction catheter below level of PBS.
    2. Using a suction catheter connected to a 1000ml pipette, 20 unfiltered research cigarettes (1 pack) were lit and “smoked” using the vacuum port at a burn rate of ~2minutes/cigarette.
    3. The optical density of the resulting PBS mixture was 2.175 at 260nm wavelength

    tung Methods

    HC14 sample obtained from normal healthy volunteer

    March 30th, 2009
    1. ~16ml of blood obtained from normal healthy volunteer (M.B.)
    2. Samples centrifuged at 1800RCF x 25min @ 22C
    3. ~12.5ml of plasma aspirated and stored at -80C
    4. ~2.5 ml of mononuclear layer aspirated and reconstituted with PBS.  Centrifuged @ 2k RPM x 10min.
    5. Cells reconstituted with 7ml EGM-2 media and cell count was 8.2082 x 10^6 cells/ml.
    6. Cells plated onto collagen I 6well plates at 3 x 10^6 cells/well x 18 wells.

    tung EPC ,

    MV29p5 cells not ready but being monitored

    March 28th, 2009

    MV29p5 cells seeded at 60k/well on 3/26/09 are not ready and exhibit much slower growth rate than MV1 seeded at the same time.  Resistances at 48hrs are:

    1. 596
    2. 903
    3. 826
    4. 874
    5. 813
    6. 934
    7. 828
    8. 35908 (faulty electrode)

    Will monitor their resistances along with MV1 in Holder B.

    tung ECIS , , ,

    MV1p9 cells ready for treatment after 48hrs

    March 28th, 2009

    Resistances for MV1p9 cells at 60k/well (seeded 3/26/09) are:

    1. 1451
    2. 1466
    3. 1433
    4. 1474
    5. 1470
    6. 1497
    7. 1477
    8. 1508

    Will perform the same treatments as previously described using 10%, 20%, & 30% CSE with and without T10 and monitor for 40hours in Holder A.

    tung ECIS , , ,

    MV20p18 cells at 70 hours

    March 26th, 2009
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  • Resistances are as follows:

    Holder A-MV20p18 at 60k cells/well

    1. 1295
    2. 1272
    3. 1296
    4. 1278
    5. 1266
    6. 1260
    7. 1272
    8. 1304

    Holder B-MV20p18 @100k/well

    1. 1370
    2. 994
    3. 1324
    4. 1392
    5. 1295
    6. 1342
    7. 1354
    8. 34043-Bad well

    Starting data collection at 1130 on 3/26/09 and continue for 24 hours.

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    1 2 3 4 5 6 7 8
    PBS 150 146 96 46 0 100 50 0
    T10 0 4 4 4 4 0 0 0
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  • 0
    0 50 100 150 50 100 150
    DMEM 100 100 100 100 100 100 100 100

    The table shows the amounts of each additive (in uL) for each well in Holder A. Wells 3 & 6 have 10% CSE. Wells 5 & 7 have 20% CSE. Wells 6 & 8 have 30% CSE. Wells 2-5 have 100uM of T10. All concentrations are based on a total of 500uL in each well. Holder B will omit column 6 and use 20% and 30% CSE in wells 6 & 7.

    tung ECIS , , , ,