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

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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
S0 CTR 0,618 32,1 64,1 4088,3 100,0 64,1
CSE 1,409 79,5 318,1 5517,0 134,9 235,7
CSE+8-p 1,201 67,1 268,4 5710,3 139,7 192,2
CSE+Bnz 0,524 26,5 105,9 4982,7 121,9 86,9
HT31 CTR 1,067 59,1 118,1 6815,0 166,7 70,9
CSE 1,181 65,9 526,9 4659,0 114,0 462,3
CSE+8-p 1,119 62,1 497,1 4882,0 119,4 416,3
CSE+Bnz 0,360 16,6 132,8 4353,0 106,5 124,8


IL8secretionD12PLATE116032010.JPG.JPG

pg/ml pg/ml pg/mL
D12 P25 aver OD Aver Il-8 Dilu alamar blue % to control Corrected
S0 CTR 0,160 4,6 9,2 8507,3 100,0 9,2
CSE 1,475 83,5 334,1 6568,7 77,2 432,8
CSE+8-p 1,473 83,4 333,5 6875,7 80,8 412,6
CSE+Bnz 0,407 19,4 77,7 5567,3 65,4 118,7
HT31 CTR 0,173 5,4 10,9 8273,7 97,3 11,2
CSE 0,838 45,3 362,2 5215,3 61,3 590,8
CSE+8-p 1,331 74,9 599,0 5988,7 70,4 850,9
CSE+Bnz 0,287 12,3 98,1 2887,0 33,9 289,2


IL8secretionD12PLATE216032010.JPG

pg/ml pg/ml pg/mL
D9 P21 aver OD Aver Il-8 Dilu alamar blue % to control Corrected
S0 CTR 0,173 5,4 10,9 10141,3 100,0 10,9
CSE 0,503 25,2 100,8 9616,0 94,8 106,3
CSE+8-p 0,502 25,1 100,6 10673,0 105,2 95,6
CSE+Bnz 0,271 11,3 45,1 11619,7 114,6 39,3
HT31 CTR 0,239 9,4 18,7 8867,3 87,4 21,4
CSE 0,591 30,5 244,0 9047,0 89,2 273,5
CSE+8-p 0,586 30,2 241,3 9436,7 93,1 259,3
CSE+Bnz 0,274 11,5 91,7 11161,3 110,1 83,3


IL8secretionD9PLATE316032010.JPG

Conclusion

  • DONE Ow..

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