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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 CSE, ECIS, MV1, T10

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 CSE, MV1, wound assay

Resistances as follows:

Holder A - MV1p10 @ 100k/well

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

Holder B - MV1p10 @ 100k/well

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

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She would never admit that she had been shunned numerous times which she found out from Tanya later.

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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	15	50	100	15	50	100
DMEM	500	496	481	446	396	485	450	400

tung ECIS CSE, ECIS, MV1, T10

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:

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DMEM	100	100	100	100	100	100	100	100

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

tung ECIS CSE, ECIS, MV1, PAR1, T10

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 ECIS, MV1, wound assay

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 CSE

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 EPC, HC14

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 CSE, ECIS, MV29, T10

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

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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 CSE, ECIS, MV1, T10

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.

Additive	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
CSE	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.

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tung ECIS CSE, ECIS, MV20, PAR1, T10