Summary
- New batch of Ht31P to use
- 16Feb2010
- Splitting cells
Materials & Methods
Materials
Stimulation
ELISA
- Supernatants collected 5March2010 (D12), 10March2010 (D12, P25) & 11March2010 (D9, P21)
- PBS 5%
- BSA 5% in PBS (20 mL per 96-wells)
- Pelikine Compact™ Human IL-8 ELISA kit
- Washing buffer (PBS + 0.005% TWEEN 20)
- IL-8 antibody
- Streptavidin conjugated to HRP
- Substrate buffer
- 1.5 sodiumacetate, resuspend in 80 mL ultrapure water
- pH 5.5
- up to 100 mL with ultrapure water
- TMB stock solution (light sensitive)
- Dilution buffer (1:5, 32 mL H2O, 8 mL undiluted buffer)
Method
- Remove medium form 24-wells plate with ASMC's grown ON in DMEM (S0)
- Rinse twice with warm PBS and remove buffer
- Add 300 μL DMEM S0 with HT31P 0 - 50 μM (see schedule below)
- Incubate 20 min.
- Prepare 25 mL DMEM S0 with 100% CSE
- Dilute 100% CSE to 30% CSE (3 mL CSE 100% + 7 mL S0)
- Add 300 μL 30% CSE to wells according to schedule below (lane C & D)
- Incubate 24 h.
Stimulation set-up
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HT31P 0 μM 1
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HT31P 10 μM 2
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HT31P 20 μM 3
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HT31P 30 μM 4
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HT31P 40 μM 5
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HT31P 50 μM 6
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A (CTR)
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B (CTR)
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C (CSE)
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D (CSE)
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CTR = control, CSE = Cigarette Smoke extract
Preparing Sandwich
- Wash coated plate 5x 200 μL PBS
- Remove PBS
- Add 200 μL BSA
- Incubate 1h @ RT and remove BSA
- Dilute samples as shown below for each 24-wells (dilution buffer (μL)/sample (μL))
24-wells (dilution buffer (μL)/sample (μL))
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1
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2
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3
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4
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5
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6
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A
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100/100
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100/100
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100/100
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100/100
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100/100
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100/100
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B
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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C
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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D
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150/50
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150/50
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150/50
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175/25
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175/25
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175/25
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- Wash of BSA with 200 μL Washing buffer 5x
- Add 100 μL of diluted sample
- Incubate ≥1h @ RT
- Wash 5x 200 μL Washing buffer
- Remove buffer
- prepare biotinylated antibody
- Add 100 μL Biotinylated antibody
- Incubate 1h @ RT
- Remove antibody
- Wash 5x 200 μL Washing buffer
- Add 100 μL streptavidin conjugated to HRP
- Incubate 25 min. @ RT
- Remove liquid
- Wash 5x 200 μL Washing buffer
- prepare TBM substrate
- 1.2 μL H2O
- 400 μL TBM stock solution
- 24 mL substrate buffer
- Add 100 μL substrate
- Incubate 30 min. @ RT
- Stop reaction, add 100 μL stopping solution
- Measure absorbance @ 450 nm
- Check duplo standard curve
- Check if values fall within standard curve (preferred)
96-wells plate ELISA
#
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01 S0
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02 S0
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03 S0
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04 Ht31
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05 Ht31
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06 Ht31
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07 S0
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08 S0
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09 S0
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10 Ht31
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11 Ht31
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12 Ht31
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A CTR
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D12 (5March2010)
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D12, P25 (10March2010)
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B CSE
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C CSE+8P
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D CSE+6Bnz
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E CTR
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D9, P21 (11March2010)
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STND 240 pg/mL
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STND 96 pg/mL
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STND 38.4 pg/mL
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STND 15.4 pg/mL
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STND 6.1 pg/mL
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STND 2.5 pg/mL
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F CSE
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STND 240 pg/mL
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STND 96 pg/mL
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STND 38.4 pg/mL
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STND 15.4 pg/mL
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STND 6.1 pg/mL
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STND 2.5 pg/mL
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G CSE+8P
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STND 1 pg/mL
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STND 0 pg/mL
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H CSE+6Bnz
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STND 1 pg/mL
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STND 0 pg/mL
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BLANC
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BLANC
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Co-immunoprecipitation
- resuspend beads prepared 15March2010
- Prepare for all donors (D9 & D12)
- Only beads (30 μL)
- Beads w. antibody (30 μL)
- Beads w. antibody (30 μL) & lysis buffer (200 μL)
- Beads w. antibody (30 μL) & sample (200 μL)
(possible also beads (30 μL) with only sample (200 μL))
Splitting cells
- D9 P23 & D12 P27 were split, one petridish to 4 (P+1)
Results
96-wells plate ELISA
#
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01 S0
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02 S0
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03 S0
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04 Ht31
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05 Ht31
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06 Ht31
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07 S0
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08 S0
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09 S0
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10 Ht31
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11 Ht31
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12 Ht31
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A CTR
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0,328
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0,555
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0,680
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1,051
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1,127
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1,024
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0,187
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0,159
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0,133
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0,156
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0,182
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0,182
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B CSE
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0,836
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1,341
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1,476
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1,188
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1,093
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1,261
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1,623
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1,367
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1,436
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0,819
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1,111
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0,856
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C CSE+8P
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1,148
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1,229
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1,227
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0,999
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1,185
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1,172
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2,237
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1,332
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1,613
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1,456
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1,158
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1,379
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D CSE+6Bnz
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0,530
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0,546
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0,496
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0,388
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0,295
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0,396
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0,458
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0,394
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0,368
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0,288
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0,284
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0,290
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E CTR
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0,175
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0,185
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0,160
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0,213
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0,268
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0,236
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1,958
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1,374
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0,709
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0,355
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0,180
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0,119
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F CSE
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0,280
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0,704
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0,525
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0,553
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0,571
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0,650
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1,815
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1,359
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0,705
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0,404
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0,192
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0,129
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G CSE+8P
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0,464
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0,516
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0,526
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0,427
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0,589
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0,741
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0,089
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0,064
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0,071
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0,073
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0,070
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0,086
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H CSE+6Bnz
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0,378
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0,220
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0,214
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1,733
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0,326
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0,222
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0,091
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0,071
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0,071
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0,013
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0,014
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0,034
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pg/ml
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pg/ml
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pg/mL
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D12
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aver OD
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Aver Il-8
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Dilu
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alamar blue
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% to control
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Corrected
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S0 |
CTR |
0,618 |
32,1 |
64,1 |
4088,3 |
100,0 |
64,1
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CSE |
1,409 |
79,5 |
318,1 |
5517,0 |
134,9 |
235,7
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CSE+8-p |
1,201 |
67,1 |
268,4 |
5710,3 |
139,7 |
192,2
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CSE+Bnz |
0,524 |
26,5 |
105,9 |
4982,7 |
121,9 |
86,9
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HT31 |
CTR |
1,067 |
59,1 |
118,1 |
6815,0 |
166,7 |
70,9
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CSE |
1,181 |
65,9 |
526,9 |
4659,0 |
114,0 |
462,3
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CSE+8-p |
1,119 |
62,1 |
497,1 |
4882,0 |
119,4 |
416,3
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CSE+Bnz |
0,360 |
16,6 |
132,8 |
4353,0 |
106,5 |
124,8
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pg/ml
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pg/ml
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pg/mL
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D12 P25
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aver OD
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Aver Il-8
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Dilu
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alamar blue
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% to control
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Corrected
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S0 |
CTR |
0,160 |
4,6 |
9,2 |
8507,3 |
100,0 |
9,2
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CSE |
1,475 |
83,5 |
334,1 |
6568,7 |
77,2 |
432,8
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CSE+8-p |
1,473 |
83,4 |
333,5 |
6875,7 |
80,8 |
412,6
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CSE+Bnz |
0,407 |
19,4 |
77,7 |
5567,3 |
65,4 |
118,7
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HT31 |
CTR |
0,173 |
5,4 |
10,9 |
8273,7 |
97,3 |
11,2
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CSE |
0,838 |
45,3 |
362,2 |
5215,3 |
61,3 |
590,8
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CSE+8-p |
1,331 |
74,9 |
599,0 |
5988,7 |
70,4 |
850,9
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CSE+Bnz |
0,287 |
12,3 |
98,1 |
2887,0 |
33,9 |
289,2
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pg/ml
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pg/ml
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pg/mL
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D9 P21
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aver OD
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Aver Il-8
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Dilu
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alamar blue
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% to control
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Corrected
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S0 |
CTR |
0,173 |
5,4 |
10,9 |
10141,3 |
100,0 |
10,9
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CSE |
0,503 |
25,2 |
100,8 |
9616,0 |
94,8 |
106,3
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CSE+8-p |
0,502 |
25,1 |
100,6 |
10673,0 |
105,2 |
95,6
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CSE+Bnz |
0,271 |
11,3 |
45,1 |
11619,7 |
114,6 |
39,3
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HT31 |
CTR |
0,239 |
9,4 |
18,7 |
8867,3 |
87,4 |
21,4
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CSE |
0,591 |
30,5 |
244,0 |
9047,0 |
89,2 |
273,5
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CSE+8-p |
0,586 |
30,2 |
241,3 |
9436,7 |
93,1 |
259,3
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CSE+Bnz |
0,274 |
11,5 |
91,7 |
11161,3 |
110,1 |
83,3
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Conclusion
Related entries
Run 4: Ht31P dose/response curve D9/D12
Run 3: Ht31/S0
- Counting cells
- Putting to S0
- Stimulating cells
- Ending stimulation & Viability
- ELISA
Same actions
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