Cell Culture Notebook

Contents

7-27-2009

• Prepare cells (HeLa) for transfection: 1,5*10⁴ cells/well; 200 µl DMEM+++/well (Ibidi)

7-28-2009

• transient transfection of HeLa cells with 3J2, 8J4 (plasmid w/ mCherry and synthetic Jet-Promotor), 8M1, 8M3 (plasmid w/ mCherry and synthetic Min-Promotor), 3, 8 (plasmids with mCherry and CMV-Promotor), positive and negative control (mGFPn1 or plasmid 6 respectively)
• Lab Tek 8 chamber allocation:

7-29-2009

• Washing (PBS) and changing medium of transfected cells.

7-30-2009

• no fluorescence with 8M1, 8M3, 8J2, 8M4 (makes sense, because we had it sequenced and there's only half of mCherry in there)
• positive control worked (GFP)

IDs created

• 30.7.1 = 3J2 - mcherry fragment + GFP
• 30.7.2 = 8M3 - mcherry fragment + GFP
• 30.7.3 = 3J2 - mcherry fragment + mcherry
• 30.7.5 = BBb integration plasmid (pcDNA5/FRT derived)
• 30.7.6 = BBb submission plasmid (pSB1A derived)
• 30.7.7 = 8M3 - mcherry fragment + mcherry

8-03-2009

• split Helas --> prepared 2x8 chambers for transient transfection (1,5x10⁴ cells/well)

8-04-2009

• transfected Hela in 8 chamber lab tek (morning; 1-8) with P.32 (CFP_emp_cytosole), P.34 (YFP_GPI_PM), P.31 (pcDNA5/FRT/GFP JeT), P.30 (pcDNA5/FRT/GFP Min), XXX (label washed off in centrifuge before - probably plasmid with mCherry and Min or Jet), P.6 (negative control; without fluorescent protein), mEGFPn1 (positive control)
• transfected Hela in 8 chamber lab tek (afternoon; 9-16) with P.38 (BFP, nucleus), P.39 (YFP, nucleus), P. 40 (mCherry, nucleus), P.42 (GFP, plasma membrane), P.43 (mCherry, plasma membrane), P.44 (GFP, mitochondria)
• changed medium of morning transfection (add 300 µl fresh medium)

8-05-2009

• changed medium of afternoon transfection from 8-4-2009
• looked at all transfections --> results: targeted proteins work (but: yellow can't be distinguished from green); Min/Jet with GFP doesn't work; XXX doesn't work either
• prepared new transfections in 96 well format: 12 wells for each cell type (HeLa --> splitted 1:10 for keeping, MCF-7, U2-OS) 200µl medium with 0,75x10⁴ cells/well

8-06-2009

• transfection of HeLa, MCF7 and U2OS (prepared 8-5-09) with p12 (mCherry, Min promoter), p33 (YFP, cytosole), p35 (CFP, plasma membrane), p36 (YFP, ER), p37 (CFP, ER), p41 (YFP, nucleus), XXX (???, label washed off in centrifuge), p6 (negative control) and eGFP-N1 (positive control) according to effectene protocol for 96-well plate

8-13-2009

• prepared transfections: 12 wells of 24-well plate with 2,5*10⁴ HeLa cells/well (originally 58*10⁴ cells/ml)

8-14-2009

• transfection of HeLa cells (8-13-09) using effectene-protocol: two samples of Jet/EGFP in pcDNA5/FRT and positive control (pEGFP-N1)

8-15-2009

• changed medium of transfected cells (DMEM+++ without indicator)
• checked transfected cells of flourescence: both transfections with jet/EGFP in pCDNA5/FRT worked (green fluorescence), positive control worked

8-19-2009

• prepared transfection of HeLa and U2-OS: 9 wells of 24-well plate per cell line , 1 ml DMEM+++ per well, 3*10⁴ cells/well

8-20-2009

• Transfections were done according to effectene protocol
• transfected plasmids

8-21-2009

• changed medium of transfected cells to remove abundant transfection complexes
• results of transfection: all transfections worked well

8-26-2009

• prepared HeLa for transfections:
  • 7 24-wells
  • 8 96-wells
  • 4 6-well
• prepared U2-OS for transfections:
  • 7 24-wells

8-27-2009

• Transfections were done according to effectene protocol
• transfected plasmids:

8-28-2009

• changed medium of transfected cells to remove abundant transfection complexes
• all transfections worked the way they were supposed to be

8-30-2009

• prepared HeLa cells for transfection: 10⁴ cells/well, 150 µl DMEM+++/well
  • 2 96-well plates (full)
  • 2 96-well plates (half)

8-31-2009

• transfection of the previously prepared cells with
  • synthetic p53 promoters (48 samples/48 controls)
  • synthetic NFκB promoters (15 samples/15 controls)
  • synthetic constitutive promoters (7 samples/7 controls)
  • synthetic "empty" promoters (10 samples/10 controls)
  • synthetic HIF promoter (32 samples/32 controls)

9-01-2009

• removed transfection complexes and changed meidum of transfected cells
• applied drugs to the cells:

• Screening results (detailed results are found here)
• All constitutive promoters were active
• "empty" promoters were not active
• Numerical evaluation of regulated (HIF, NFκB, p53) promoters: From 0 = no fluorescnence to 3 = strong fluorescence can be found in Results 9-1-2009
  • HIF: induction time was too short
  • p53: most cells were dead, as the cells were left in Camptothecine for too long

• HeLa cells don't survive in minimal medium over night! -> use MCF-7 instead

9-02-2009

• prepared HeLa cells for transfection:
  • 24 6-wells with 10⁵ cells/well
  • 144 96-wells with 10⁴ cells/well
• 6x10⁶ cells for RNA extraction isolated

9-03-2009

• transfected HeLa according to effectene protocol:
• transfected plasmids:
  • HIF S 3-20,23,24 ; HIF L 4,6-9,11,16,17,19,20,21,23 → Because bad choice of timepoint: Wait ~5h
  • p53 S 8, 13, 22, 23, 24; p53 L 1, 3, 9, 11, 13, 17, 18
  • P7_CMV_mCherry

9-09-2009

• transfection for p53 & NFκB screening:
  • 2x28 96-wells of MCF-7 for p53
  • 2x34 96-wells of U2-OS for NFκB

9-10-2009

• transfections for FACS (see here for description of used plasmids):
  • triplicates of the following for each HeLa, U2-OS, MCF-7: p6 (negative control), p43 (mCherry, CMV promoter), p55 (GFP, CMV promoter), p31 (GFP, JeT promoter), p48 (GFP, CMVcoreJeTprox promoter), p55&p43 (1:2), p31&p43 (1:2), p48&p43 (1:2)
  • triplicates of the following (c=constitutive) for Hela and MCF-7: constitutives --> cL1&p43 (1:2), cL4&p43 (1:2), cL12&p43 (1:2), cS5&p43 (1:2), cS10&p43 (1:2), cS4&p43 (1:2)
• transfection for microscopy:
  • one replicate of the following, each for Hela and MCF-7 (with coverslips): cL1&p43 (1:2), cL4&p43 (1:2), cL12&p43 (1:2), cS5&p43 (1:2), cS10&p43 (1:2), cS4&p43 (1:2), p31&p43 (1:2)

• prepared 0,45x10⁶ cells (regular HeLa) for genomic DNA extraction --> didn't work (0,9 ng/µl)
• changed medium of yesterdays p53, NFκB transfections
  • p53: minimal medium & minimal medium + 1:1000 CPT (stock: 2 mM)
  • NFkB: minimal medium & minimal medium + 1:1000 TNF-a (stock: 2,5 mM)

==> SCREENING of p53 & NFκB

9-11-2009

• changed medium on FACS and microscopy plates (from 9-10): regular DMEM+++ for FACS, indicator-free DMEM+++ for microscopy
• constitutive promoters not active!?! --> redo
• test tranfected one 24-well of MCF-7 with p38 (BFP, nucleus), p44 (GFP, mitochondria), p43 (mCherry, plasma membrane) for Lipofectamine testing

9-12-2009

• prepared 3x48 wells (96-well format) with 10⁴ cells (MCF-7) for HIF promoter testing +2 wells for JeT
• prepared 24 wells (96-well format) with 10⁴ cells (HeLa) for repeat of constitutive promoter testing

9-13-2009

• transfected HIF S1-S24 & HIF L1-L24
• transfected constitutive promoters (cS1-24, cL1-24; w/o p43)

9-14-2009

• changed medium of constitutive stuff (DMEM+++) --> 80% of constructs show fluorescence --> problem with co-transfection?!?
• changed medium on HIF stuff: two replicates minimal medium (Krebs-Henseleit) & one replicate minimal medium + Everolimus (rapamycine like --> HIF inhibitor; diluted 1:500 000)
• put one replicate with minimal medium in HIF bag --> left for 8 hours; tablet not really white; more light blue

==> SCREENING --> HIF induction does not seem to work; also barely any difference between negative control and inhibited (Everolimus) samples

• prepared 3x34 wells of U2-OS for repeat of NFκB testing (96-well format)
• prepared 16 wells of HeLa (96-well format) for testing of mCherry ligation in p7 (unmethylated p6 -> without any construct) and p7/JeT + 4 wells for testing Douaa's mutagenesis of p33 (YFP, cytosole), p35 (CFP, plasma membrane), p37 (CFP, ER), p37ras
• prepared 3x30 wells of HeLa for testing of constitutive promoters using FACS (96-well format!!)

9-15-2009

• transfection (Effectene) of 3x34 96-wells of U2-OS with NFkB constructs
• transfection (Effectene) of ligations and Douaas stuff for testing
• transfection (Effectene) of constitutive promoters (cS1-12, cL1-12, #5, 13, 16, 21 of the p53 promoters --> seemed to be constitutive, p6 --> negative control, p31 (GFP, JeT promoter) --> positive control): everything except p6 --> co-transfection with p43 (construct:mCherry --> 1:2)
• prepared 6 24-wells of HeLa with coverslips for transfection with Lipofectamine

9-16-2009

• changed medium of yesterday's transfections:
  • A1/A2 --> regular DMEM+++ --> no fluorescence
  • Douaa's mutagenesized constructs --> regular DMEM+++ --> p33: fluorescence in red, even stronger in green channel; p35: very week in green; p37, p37ras: stronger in green ==> p33, p37, p37ras okay, p35?!
  • constitutive promoters for FACS measurement (96-well format) --> looks bad again; FACS results show no green fluorescence, only red --> retransformed p55 (GFP, CMV promoter), p31 (GFP, JeT promoter), p43 (mCherry); change ratio of constructs: p43 to 2:1 in future
  • dye-free DMEM+++, minimal medium, minimal medium + TNF%alpha; (1:1000) for U2-OS (NFkB)

==> SCREENING (microscopy and TECAN --> approx. 8 promising promoters)

• transfection of HeLa for microscopy (6 24-wells): p55&p43 (2:1), p31&p43 (2:1), p48&p43 (2:1), p31&p43 (2:3), S10&p43? (2:3), L9&p43? (2:3)
• transformed and plated constitutive promoters again (S1-12, L1-12)

• preparation of cells:
  • 3 24-wells with coverslips of MCF-7
  • 3 24-wells with coverslips of U2-OS
  • 2x96 + 1x48 wells (96-well format) of MCF-7 for HIF and p53 screening

• made 50 µl aliquots of DFO (induces HIF --> use in combination with hypoxia bag for 24 to 48 hours)

9-17-2009

• transfections:
  • 3 24-wells of MCF-7 with p55&p43 (2:1), p31&p43 (2:1), p48&p43 (2:1) (Lipofectamine)
  • 3 24-wells of U2-OS with p55&p57 (2:1), p31&p57 (2:1), p48&p57 (2:1) (Lipofectamine)
  • p53 (Effectene): 3x28 wells on one plate + 2xp6 + 2xp31
  • HIF (Effectene): 2x48 wells on one plate; 1x48 wells + 1xp6 on second plate (hypoxia bag)

• changed medium (dye-free DMEM+++) on 6 24-wells for microscopy

9-20-2009

• changed medium of transfected cells (morning)

9-21-2009

• prepared 12 replicates of 1,5*10⁵ cells per promoter (Jet/CMV)
• prepared HeLa for constitutive promoter measurement: 24 promoters, 3 controls (3 replicates for each)
• prepared HeLa for AHR screening: 96 wells

9-22-2009

• prepared cells for SREBP-, PPARγ- and estrogen screening:
  • SREBP: 9 samples for Hela
  • PPARγ: 7 samples for U2-OS
  • Estrogen: 9 samples for MCF-7
• prepared HeLa cells for constitutiv promoter meassurement: 81 96-wells

9-23-2009

• transfection of constitutive promoters in HeLa: 3xp6, 3xp31/p6 (2:1), 3x24 constitutive promoters/p6 (2:1)
• transfection of
  • AHR: p6, p31, 45xAHR, 1x CYP (natural promoter)
  • SREBP: p6, p31, 18xSREBP
  • PPARy: p6, p31,14xPPARγ
  • Estradiol: p6, p31,18x Estradiol
• prepared MCF-7 for p53 transfections

9-24-2009

• transfection of MCF-7:6xp6, 6xp31/p6 (2:1), 6xp53 promoters (S2, 5, 8, 9 ,1; L1, 4, 17, 18)
• induce yesterdays transfections:
  • SREBP:
    • inactivating medium: DMEM+++, LDL, Cholesterol (both 10µg/ml)
    • activating medium: DMEM, 0,5% LDS, 1% glutamin, 1% penstrep
  • Estrogen: keep one day in FCS free DMEM++
• AHR: induce with TCCD 1:3000 in fullmedium
• PPARγ: not induced because drug not available

9-25-2009

• screening
  • AHR at 2 pm
  • estrogen at 5 pm
• induced p53: every two minutes 3 replicates of one promoter induced

9-26-2009

• prepared cells for transfections:
  • Hela
    • 96 wells for AHR
    • 24 wells for SREBP
  • U2-OS: 24 96-wells for PPARY

9-27-2009

• prepared cells for transfection SREBP, PPARγ, Estrogen and AHR again

9-28-2009

• prepared cells for transfections:
  • HeLa: 81 96-wells for constitutive promoters
• SREBP screening
• transfected AHR/CYP, SREBP, Estrogen, PPARγ

9-29-2009

• induced yesterdays transfections:
  • SREBP:
    • inactivating medium: DMEM+++, LDL, Cholesterol (both 10µg/ml)
    • activating medium: DMEM, 0,5% LDS, 1% glutamin, 1% penstrep
  • Estrogen: keep one day in FCS free DMEM++ (add Estradiol tomorrow (9-30))
  • AHR: induce with TCCD 1:3000 in fullmedium -> screen 9-30 at 8 am (after 18 h)
  • PPARγ: Thiazolidinedione 1:100 000 -> screen 10-1 at 1.30 pm (after approx. 54 h)
• transfected the following:
  • triplicates of constitutive promoters (24) in HeLa for FACS + p31, p6 (all except p6 as cotransfections with p57)
• prepared the following cells for transfection on 9-30-2009:
  • HeLa for constitutives (FACS)
  • U2-OS: 90 96-wells for NFκB promoters

9-30-2009

• induced:
  • put β-Estradiol on MCF-7 at 8 am -> TECAN 10-1 at 8 am (after 24 h)
• transfected the following:
  • triplicates of NFκB promoters (12 good ones + p31, p6), each induced and not induced (in total 6 wells for each promoter) for FACS (cotransfection with p57)
  • triplicates of 24 constitutive promoters (+ controls) for FACS (cotransfections with p57) on 10-1
• prepared cells:
  • U2-OS for NFκB (FACS)
  • 24-wells HeLa with coverslips for microscopy
  • HeLa for SREBP (TECAN)
  • MCF-7 for HIF (FACS)

10-01-2009

• induced:
  • 7 am: NFκB with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm
• transfected:
  • triplicates of HIF in MCF-7 (8 candidates + controls), each uninduced, inhibited (Everolimus) and induced (HIF bag + DFO)
  • triplicates of NFkB promoters (12 good ones + p31, p6), each induced and not induced (in total 6 wells for each promoter) for FACS
  • SREBP -> three wells for each promoter (uninduced, inhibited, induced)
• prepared cells:
  • MCF-7 for p53 (FACS)
  • U2-OS for PPARγ (TECAN)
  • MCF-7 for Estrogen (TECAN)

10-02-2009

• induced:
  • 7 am: nfk-b with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm
  • 10 am: HIF for FACS on 10-3-09 at 10 am
  • 9 am: change media of SREBP transfection from 10-1 (uninduced -> full medium, inhibiting -> DMEM+++ with LDL & Cholesterol, inducing -> DMEM++/without FCS with 0,5% LDS instead)
  • 8.50 am: put 1% HPCD (in DMEM++ with 0,5% LDS) for three hours on SREBP transfection from 9-28; leave for three hours, remove & put DMEM++ with 0,5% LDS back on the cells -> TECAN every 1,5 hours for three hours
  • 7 pm: CPT on p53 -> FACS 10-3 at 7 am
• transfections:
  • triplicates of p53 promoters (9 candidate + p31, p6), each uninduced, inhibited and induced (in total 9 wells for each promoter) for FACS (cotransfections with p57)
  • PPARy -> two wells for each promoter (induced, uninduced)
  • Estrogen -> two wells for each promoter (induced, uninduced)
• prepared cells:
  • U2-OS for NFκB

10-03-2009

• induced:
  • PPARy for TECAN on 10-5
  • DMEM++ on Estrogen stuff
• transfections:
  • NFκB (6 promoters + controls) for FACS
• prepared cells:
  • HeLa for constitutive promoters
  • HeLa for standard
  • HeLa for AHR (9 promoters + controls)
  • 24-wells HeLa with coverslips for microscopy

10-04-2009

• induced:
  • β-Estradiol on Estrogen stuff -> TECAN 10-5 after 24 h
  • 8.30 am: NFκB with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 6.30 pm
• transfected:
  • 24 constitutive promoters + controls (in HeLa) for FACS
  • standard in HeLa for FACS
  • AHR (9 promoters + controls) for FACS
  • microscopy???

10-05-2009

• induced:
  • 3 pm: AHR (Benzo-a-pyren) for FACS on 10-6 at 9 am
  • 9 am: put 1% HPCD (in DMEM++ with 0,5% LDS) for three hours on SREBP transfection from 10-1; leave for three hours, remove & put DMEM++ with 0,5% LDS back on the cells -> TECAN every 1,5 hours for three hours
• prepared cells:
  • HeLa for SREBP for FACS
  • MCF-7 for Estrogen for FACS
  • 12 6-wells U2-OS
  • 8 24-wells HeLa with coverslips for microscopy
  • U2-OS for NFkB for FACS
  • U2-OS for NFkB (5 promoters + controls) for microscopy with H. Erfle's group
  • 2x8 96-wells U2-OS for FACS test

10-06-2009

• transfections:
  • Estrogen in MCF-7 for FACS (3 promoters)
  • Nathan's ibidi (MCF-7) with NFκB promoters (p31, NIL12, NIIS1, NIIL10 -> two transfections each = 8 in total)
  • NFκB (6 promoters + controls in U2-OS) for FACS
  • NFκB (5 promoters + controls) for microscopy with H. Erfle's group
  • 8xp6 and 8xp31/p6 (2:1) for FACS test
  • SREBP (HeLa) for FACS
• prepared cells:
  • HeLa for SREBP for TECAN
  • 4 24-wells HeLa for FACS test
  • 4 24-wells MCF-7 for FACS test
  • U2-OS for PPARγ once for TECAN, once for FACS
  • U2-OS and MCF-7 for NFκB for TECAN
  • MCF-7 for p53 for TECAN

10-07-2009

• induced:
  • NFκB in ibidi with TNFα (4.50 am; used DMEM+++ instead of DMEM++ -> TNFα probably inactivated -> "reinduced" at 8 am -> ready for fixation at 6.15 pm)
  • NFκB in 96-well for FACS (4.15 am -> FACS at 2.15 pm)
  • NFκB (5 promoters + controls) for microscopy with H. Erfle's group at 5.20 am -> fix with Pfa at 3.20 pm; microscopy appointment tomorrow 10-8 at 3 pm
  • put minimal medium on Estrogen stuff
  • SREBP (HeLa) for FACS: put medium on cells (full medium, inactivating & activating medium)
• transfections:
  • SREBP (HeLa) for TECAN
  • 2xp6 and 2xp31/p6 (2:1) in HeLa for FACS test
  • 2xp6 and 2xp31/p6 (2:1) in MCF-7 for FACS test
  • PPARy (4 promoters + controls) in U2-OS once for FACS, once for TECAN
  • NFκB (2 promoters + controls) in U2-OS and MCF-7 for TECAN
  • p53 (4 promoters (realized later, that they were chosen wrongly from preliminary results -> redo) + controls
  • NFκB (NIIL10) in 24-wells for microscopy (one replicate for induction, one to leave uninduced
• prepared cells:
  • HeLa for TECAN of natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP, c-Jun)
  • HeLa for FACS of const (6 promoters) and standard (24 wells)
  • HeLa in 24-wells with coverslips for microscopy

10-08-2009

• induced:
  • SREBP for TECAN: changed media (full medium, inactivating, activating) at 9 am
  • put Estradiol on Estrogen stuff at 8 am -> FACS 10-9 at 8 am
  • PPARγ for FACS and TECAN at 7.45 am -> FACS/TECAN 10-10 at 4 pm
  • p53 for TECAN at 7.30 am (minimal medium, activating, inactivating) -> TECAN at 7.30 pm
  • NFκB (U2-OS and MCF-7) for TECAN at 7.30 am -> TECAN at 5.30 pm
  • NFκB (U2-OS in 24-wells for microscopy) at 7.30 am -> fixed for microscopy after 1 h and after 10 h (induced and uninduced for each time point)
• transfections:
  • natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP, c-Jun) for TECAN
  • const and standard for FACS in HeLa -> FACS 10-09 at 1 pm (put on TECAN before FACS measurement)
  • 24-wells: p31, p55, p48 each cotransfected with p57
• prepared cells:
  • HeLa for natural promoters (CYP, c-jun on one plate) for FACS
  • HeLa for natural promoters (2xSREBP) for FACS
  • U2-OS for cross induction of good NFκB promoter with PPARγ drug

10-09-2009

• induce:
  • natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP -> change medium, c-Jun -> put DMEM++ on cells) for TECAN
  • HPCD on SREBP for FACSat 9 am -> removed at noon -> FACS at 3 pm
• transfections:
  • natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP) for FACS
  • natural promoters (c-Jun, CYP -> just change full medium) for FACS
  • NFκB (1 promoter -> NIIL10 + controls) for cross induction with PPARγ drug
• prepared cells:
  • HeLa for constitutives and standard

10-10-2009

• induce:
  • SREBP for TECAN: HPCD at 9.30 am, remove at 12.30 pm -> TECAN at 3.30 pm
  • c-jun: EGF (diluted 1:2000 in DMEM++) on cells for three hours (add 10 am, remove 1 pm) -> TECAN at 3 pm
  • natural promoters (c-Jun -> add DMEM++ at 10 am, CYP -> just change to fresh full medium at 10 am; induce with Benzo-a-pyren at 7 pm) for FACS on 10-11 at 1 pm
  • cross induction of NFκB promoter (NIIL10, p31, p6): one triplicate each with DMEM++ +PPARγ drug, one triplicate each with just DMEM++, one triplicate each with minimal medium, one triplicate each with full medium -> FACS on 10-12 at 6 pm
  • natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP) for FACS: change medium
• transfections:
  • HeLa for constitutives and standard -> FACS 10-11
• prepared cells:
  • HeLa, MCF-7 & U2-OS for standard (on one plate)
  • HeLa in black plates for const (for microscopy in H. Erfle's group)
  • MCF-7 for p53 for TECAN
  • MCF-7 for p53 for FACS
  • ibidi (8 wells) with 1,5x10^4 cells/well for time lapse fluorescence movie of NFκB induction

10-11-2009

• induce:
  • EGF on c-Jun at 8 am -> FACS at 1 pm (one plate with CYP -> induction yesterday)
• transfection:
  • standard in HeLa, MCF-7, U2-OS -> FACS on 10-12 at 6 pm
  • const in HeLa (for microscopy in H. Erfle's group)
  • p53 in MCF-7 for TECAN
  • p53 in MCF-7 for FACS
  • for live video of NFκB induction: cotransfection of plasma membran localized mCherry with JeT promoter and non localized GFP with NIIL10 (self-synthesized NFkB promoter)
• prepared cells:
  • 36 6-wells for time course measurement of NIIL10 (6 replicates at four time points (induced) + six replicates at two time points (uninduced))
  • ibidi with U2-OS for measurement of NFκB activation by fluorescent construct that localizes to nucleus
  • MCF-7 for natural promoter (HSP70 -> heat shock) -> on two plates
  • 12 24-wells of U2-OS with coverslips for microscopy

10-12-2009

• induce:
  • p53 in MCF-7 once for FACS, once for TECAN (CPT for activation, Pifithrin-α for inactivation, minimal medium as reference) at 7.25 am
  • HPCD on natural promoters for FACS at 11.30 am -> remove at 2.30 pm -> FACS at 5.30 pm
  • HPCD on natural promoters for TECAN at 11.30 am -> remove at 2.30 pm -> TECAN at 5.30 pm
• transfections:
  • NIIL10 in 30* 6-wells for time course measurement
  • HSP70 (natural heatshock promoter) in MCF-7 for TECAN: triplicates of HSP70, p31, p6
  • NIIL10 in U2-OS in 24-wells with coverslips for microscopy after 1 h and 10 h: 6* cotransfections for each NIIL10/p57, p31/p57 (ratio 2:1)
  • NIIL10 in U2-OS (8*ibidi) with 4* cotransfections for each NIIL10/p57, p31/p57 (ratio 2:1)
  • p53 in MCF-7 in 96-wells: 4* triplicates of each pS9 and JeT, 4*p6; triplicates of each pS9/const AMPK, pS9/dn AMPK, p31/const AMPK, p31/dn AMPK,1* each p6/const AMPK, p6/dn AMPK
• cells:
  • HeLa for measurement of different time points of CMV and JeT

10-13-2009

• induce:
  • 6-wells with NIIL10 for time course of NFκB induction -> induction: 7.45 am, freeze cells (6 induced and 6 non-induced replicates after 1 h, 6 induced replicates after 6 h and 9,5 h, 6 induced and 6 non-induced replicates after 11,75 h)
  • coverslips with NIIL10 for microscopy
• prepared cells for transfections:
  • U2-OS (18* 96-wells) for NFκ-B
  • U2-OS (24* 96-wells)for standard plate
  • U2-OS (8* ibidi) for NFκ-B
  • MCF-7 (24* 96-wells)standard plate
• transfections (morning):
  • HeLa: three triplicates of CMV/p57, JeT/p57, p6 for medium depend measurement of constitutive promoters
  • HeLa: two triplicates of CMV/p57, JeT/p57, p6 for time measurement of constitutive promoters
• changed medium of transfections triplicate of CMV/p57, JeT/p57, p6 with (afternoon)
  • DMEM+++ -> FACS 10-14 (20 h) & 10-15 (50 h)
  • DMEM++ -> FACS 10-14
  • DMEM++ with everolimus -> FACS 10-14

10-14-2009

• induce: 7.30 am U2-OS (8* ibidi) NFκ-B with TNFα
• transfections:
  • U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57
  • U2-OS and MCF-7 (each 24* 96-wells) each with triplicates of p6, p6/p31, p6/p48 p6/p55, p6/ p57, p57/p31, p57/p48, p57/p55 (ratio of cotransfections 1:2) -> FACS 10-15
• prepared cells:
  • U2-OS (8* ibidi) for NIIL10/JeT cotransfections

10-15-2009

• induced U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57 at 8 am -> FACS at 6 pm
• transfections:
  • U2-OS (8* ibidi) for NIIL10/JeT cotransfections (ratio 2:1)
• prepared cells for sequences ordered from Geneart:
  • 3x60 96-wells HeLa on black plates for a maximum of 13 HB_SREBPI responsive promoters (predicted with HEARTBEAT)
  • 2x60 96-wells MCF-7 on black plates for a maximum of 11 HB_VDRI responsive promoters (predicted with HEARTBEAT)

10-16-2009

• cloning did not work for all the sequences
• transfected:
  • HB_SREBPI (9 wells each) in HeLa: p6, p31/p57, clones 2, 3, 4, 7, 8, 9 and 11 (each cotransfected with p57 at a 1:2 ratio)
  • HB_VDRI (6 wells each) in MCF-7: p6, p31/p57, clones 15?, 16, 18, 20, 21, 22, 23, 24, 25 (each cotransfected with p57 at a 1:2 ratio)

10-17-2009

• induction:
  • HB_SREBPI in HeLa: full medium (DMEM+++), inactivating medium (DMEM+++ +LDL +Cholesterol), activating medium (DMEM++ +LDS instead of FCS) -> HPCD and FACS: 10-20
  • HB_VDRI in MCF-7: 10^-7M Vitamine D3 -> TECAN every 24 h; FACS 10-21

10-18-2009

• cells:
  • 2x60 96-wells MCF-7 for HB_VDRII

10-19-2009

• cells:
  • 2x60 96-wells HeLa for HB_SREBPII
  • 1 24-well of HeLa with coverslip to test if transfection also works in submission plasmid (pSB1A3, BioBricked CMV, mCherry, PM localization signal -> p53_mCherry_PM)
• transfections:
  • HB_VDRII in MCF-7: 6 wells of clones 16, 18, 20, 21, 22, 23, 24, 25; 12 wells of clones 15, 17, 19, p6, p31/p57 (all clones cotransfected as usual)

10-20-2009

• induce:
  • HB_VDRII in MCF-7: those clones with 12 replicates and controls -> 10^-7M and 10^-9M Vitamine D3, others (6 replicates) -> 10^-9M Vitamine D3
• cells:
  • 2x60 wells HeLa for HB_SREBPIII
  • 2x60 wells MCF-7 for HB_VDRIII
• transfections:
  • HB_SREBPII in HeLa: 9 wells of clones 1, 2, 3, 4, 7, 8, 9, 11 and 13; 18 wells of p6 and p31/57 (one set of controls for each plate)
  • 24-well (HeLa) with p53_mCherry_PM (Lipofectamine)

10-21-2009

• induce:
  • HB_SREBPII in HeLa: full medium (DMEM+++), inactivating medium (DMEM+++ +LDL +Cholesterol), activating medium (DMEM++ +LDS instead of FCS) -> HPCD and FACS: 10-24
• transfections:
  • HB_SREBPIII: 9 wells of clones 1, 2, 3, 4, 7, 8, 9, 11, 13; 18 wells of p6 and p31/57 (one set of controls for each plate)
  • HB_VDRIII: 9 wells (for two concentrations: 10^-7M and 10^-9M of Vitamine D3) of clones 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25