User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/16

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ELISA & hTERT stimulation Main project page
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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

Stimulation hTERT cells

  • 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
HT31P
0 μM
1
HT31P
10 μM
2
HT31P
20 μM
3
HT31P
30 μM
4
HT31P
40 μM
5
HT31P
50 μM
6
A
(CTR)
B
(CTR)
C
(CSE)
D
(CSE)

CTR = control, CSE = Cigarette Smoke extract

ELISA

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))
1 2 3 4 5 6
A 100/100 100/100 100/100 100/100 100/100 100/100
B 150/50 150/50 150/50 175/25 175/25 175/25
C 150/50 150/50 150/50 175/25 175/25 175/25
D 150/50 150/50 150/50 175/25 175/25 175/25


  • 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
    • It will turn yellow
  • Measure absorbance @ 450 nm
    • Check duplo standard curve
    • Check if values fall within standard curve (preferred)
96-wells plate ELISA
#
01
S0
02
S0
03
S0
04
Ht31
05
Ht31
06
Ht31
07
S0
08
S0
09
S0
10
Ht31
11
Ht31
12
Ht31
A
CTR
D12 (5March2010) D12, P25 (10March2010)
B
CSE
C
CSE+8P
D
CSE+6Bnz
E
CTR
D9, P21 (11March2010) STND 240 pg/mL STND 96 pg/mL STND 38.4 pg/mL STND 15.4 pg/mL STND 6.1 pg/mL STND 2.5 pg/mL
F
CSE
STND 240 pg/mL STND 96 pg/mL STND 38.4 pg/mL STND 15.4 pg/mL STND 6.1 pg/mL STND 2.5 pg/mL
G
CSE+8P
STND 1 pg/mL STND 0 pg/mL
H
CSE+6Bnz
STND 1 pg/mL STND 0 pg/mL BLANC BLANC

Co-immunoprecipitation

  • resuspend beads prepared 15March2010
  • Prepare for all donors (D9 & D12)
  1. Only beads (30 μL)
  2. Beads w. antibody (30 μL)
  3. Beads w. antibody (30 μL) & lysis buffer (200 μL)
  4. Beads w. antibody (30 μL) & sample (200 μL)

(possible also beads (30 μL) with only sample (200 μL))

  • Incubate ON @ °C

Splitting cells

  • D9 P23 & D12 P27 were split, one petridish to 4 (P+1)

Results

96-wells plate ELISA
#
01
S0
02
S0
03
S0
04
Ht31
05
Ht31
06
Ht31
07
S0
08
S0
09
S0
10
Ht31
11
Ht31
12
Ht31
A
CTR
0,328 0,555 0,680 1,051 1,127 1,024 0,187 0,159 0,133 0,156 0,182 0,182
B
CSE
0,836 1,341 1,476 1,188 1,093 1,261 1,623 1,367 1,436 0,819 1,111 0,856
C
CSE+8P
1,148 1,229 1,227 0,999 1,185 1,172 2,237 1,332 1,613 1,456 1,158 1,379
D
CSE+6Bnz
0,530 0,546 0,496 0,388 0,295 0,396 0,458 0,394 0,368 0,288 0,284 0,290
E
CTR
0,175 0,185 0,160 0,213 0,268 0,236 1,958 1,374 0,709 0,355 0,180 0,119
F
CSE
0,280 0,704 0,525 0,553 0,571 0,650 1,815 1,359 0,705 0,404 0,192 0,129
G
CSE+8P
0,464 0,516 0,526 0,427 0,589 0,741 0,089 0,064 0,071 0,073 0,070 0,086
H
CSE+6Bnz
0,378 0,220 0,214 1,733 0,326 0,222 0,091 0,071 0,071 0,013 0,014 0,034


pg/ml pg/ml pg/mL
D12 aver OD Aver Il-8 Dilu alamar blue % to control Corrected
S0CTR0,61832,164,14088,3100,064,1
CSE1,40979,5318,15517,0134,9235,7
CSE+8-p1,20167,1268,45710,3139,7192,2
CSE+Bnz0,52426,5105,94982,7121,986,9
HT31CTR1,06759,1118,16815,0166,770,9
CSE1,18165,9526,94659,0114,0462,3
CSE+8-p1,11962,1497,14882,0119,4416,3
CSE+Bnz0,36016,6132,84353,0106,5124,8


Image:IL8secretionD12PLATE116032010.JPG.JPG

pg/ml pg/ml pg/mL
D12 P25 aver OD Aver Il-8 Dilu alamar blue % to control Corrected
S0CTR0,1604,69,28507,3100,09,2
CSE1,47583,5334,16568,777,2432,8
CSE+8-p1,47383,4333,56875,780,8412,6
CSE+Bnz0,40719,477,75567,365,4118,7
HT31CTR0,1735,410,98273,797,311,2
CSE0,83845,3362,25215,361,3590,8
CSE+8-p1,33174,9599,05988,770,4850,9
CSE+Bnz0,28712,398,12887,033,9289,2


Image:IL8secretionD12PLATE216032010.JPG

pg/ml pg/ml pg/mL
D9 P21 aver OD Aver Il-8 Dilu alamar blue % to control Corrected
S0CTR0,1735,410,910141,3100,010,9
CSE0,50325,2100,89616,094,8106,3
CSE+8-p0,50225,1100,610673,0105,295,6
CSE+Bnz0,27111,345,111619,7114,639,3
HT31CTR0,2399,418,78867,387,421,4
CSE0,59130,5244,09047,089,2273,5
CSE+8-p0,58630,2241,39436,793,1259,3
CSE+Bnz0,27411,591,711161,3110,183,3


Image:IL8secretionD9PLATE316032010.JPG

Conclusion

  • DONE Ow..

Related entries

Run 4: Ht31P dose/response curve D9/D12

Run 3: Ht31/S0

Same actions

Attachment

Excel hTERT D12

Excel hTERT D12 P25

Excel hTERT D9 P21


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