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

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 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] ELISA & hTERT stimulation
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


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Summary

 * New batch of Ht31P to use
 * 16Feb2010
 * Splitting cells

Materials
Stimulation ELISA
 * hTERT (Airway smooth muscle cells, ASMC's) (D12 P27/D9 P32; split 12Feb2010, put to S0 15Feb2010)
 * InCELLect™ St-Ht31P Control Peptide
 * Warm DMEM (S0)
 * Warm PBS
 * DMEM S0
 * 500 mL DMEM
 * 3 mL Fungizone
 * 10 mL PenStrep
 * 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)
 * 30 mg TMB
 * 5 mL DMSO
 * Dilution buffer (1:5, 32 mL H2O, 8 mL undiluted buffer)

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.

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))
 * 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
 * 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
 * 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)
 * Check duplo standard curve
 * Check if values fall within standard curve (preferred)

Co-immunoprecipitation
(possible also beads (30 μL) with only sample (200 μL))
 * 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)
 * Incubate ON @ °C

Splitting cells

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

Results
     

Conclusion

 * ✅ Ow..

Run 4: Ht31P dose/response curve D9/D12

 * Splitting cells 12March2010
 * Cells put to S0 15March2010

Run 3: Ht31/S0

 * Counting cells
 * 26Feb2010 D9/D12
 * 03March2010 D9 (P21)/D12 (P25)
 * Putting to S0
 * 03March D9/D12
 * 08March2010 D12 (P25)
 * 09March2010 D9 (P21)
 * Stimulating cells
 * 04March2010 D9 (samples lost)/D12
 * 09March2010 D12 (P25)
 * 10March2010 D9 (P21)
 * Ending stimulation & Viability
 * 05March2010 D12
 * 10March2010 D12 (P25)
 * 11March2010 D9 (P21)
 * ELISA
 * 16March2010 D12, D12 (P25) & D9 (P21)

Same actions

 * Cell stimulation

Attachment
   [[Media:HTERT_Donor_12_ht31_160310.xls| Excel hTERT D12]]

  </a> [[Media:HTERT_donor_12_P25_160310.xls| Excel hTERT D12 P25]]

 <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> [[Media:HTERT_Donor_9_P21_160310.xls| Excel hTERT D9 P21]]


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