Bridges Lab Protocols - User contributions [en]

Differentiation of MEFs

2013-12-23T20:02:34Z

Nporitsa:

==Protocol==
Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
==Strains==
Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
==Source==
DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
==Papers==
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

==Materials==
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T20:02:21Z

Nporitsa:

==Protocol== Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
==Strains== Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
==Source== DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
==Papers==
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

==Materials==
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T20:02:05Z

Nporitsa:

==Protocol==Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
==Strains==Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
==Source==DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
==Papers==
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

==Materials==
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T20:01:42Z

Nporitsa:

'''==Protocol=='''Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
'''==Strains==''' Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
'''==Source==''' DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
==Papers==
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

==Materials==
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T20:01:18Z

Nporitsa:

'''==Protocol=='''Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
'''==Strains==''' Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
'''==Source==''' DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
'''==Papers=='''
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

==Materials==
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T19:58:50Z

Nporitsa: /* Induction of MEF differentiation */

Protocol: Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
Strains: Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
Source: DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
Papers:
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

Materials
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
# At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off). '''Note1:''' Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
# Protein lysate and RNA extraction is taken at day 4 (D3). '''Note2:''' In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection. '''Note3:''' A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of MEFs

2013-12-23T19:54:18Z

Nporitsa: Created new page

Protocol: Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
Strains: Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
Source: DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
Papers:
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

Materials
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

==Induction of MEF differentiation==
# Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
# On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
3. At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).
• Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
4. Protein lysate and RNA extraction is taken at day 4 (D3).
• Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.
• Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Wiki new protocols

2013-12-23T19:41:25Z

Nporitsa: Replaced content with "\"

Wiki new protocols

2013-12-23T19:37:22Z

Nporitsa: Differentiation of Tsc2 MEFs

Protocol: Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
Strains: Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
Source: DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
Papers:
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

Materials
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

Induction of MEF differentiation:
1. Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
2. On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
3. At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).
• Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
4. Protein lysate and RNA extraction is taken at day 4 (D3).
• Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.
• Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.

Differentiation of 3T3-L1 Cells

2013-12-23T19:34:37Z

Nporitsa: /* Reference */

Paraffin Embedding of Tissue Samples

2013-12-18T10:51:27Z

Nporitsa:

== Protocol ==
#After harvesting tissue from animal, place into pathology cassette and fix in 4% formaldehyde, 4% paraformaldehyde or 4% formalin O/N
#After fixation, begin dehydrating the tissue in 70% Ethanol
#On the day of planned embedding, wash sample with 100% Ethanol 3 times for 30 minutes.
##While washing in 100% Ethanol, start the paraffin wax machine (Link Building, Room 311)
#After 100% Ethanol washes, Wash 3 times in Xylenes
#Wash samples in a beaker filled with wax at 58-60C, 3 times for 15 mins
#Immediately before embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with wax
#Take your sample and orient it on top of the base wax as desired
#Place pathology cassette on top of the mold and fill with wax covering the mold
#Place mold with sample on "Cold Side" of embedding station
##Wax will solidify in 10-15 minutes
#Remove wax embedded sample from the mold (should just slip out)
#Store at room temperature until prepped to section

Protocol Edited from: FFPE SOP - MKM and SR for the Williams Lab

1. Collect tissue
a. Immediately place in labeled cassette in 10% buffered formalin
b. Place in labeled cassette on dry ice prior to storage at -80C

2. Fix in 10% buffered formalin
a. NOTE: for 3 mm mouse brain sections NEVER exceed 24H in formalin

3. Tissue processing
a. 70% EtOH (30 min) → tissues can be kept in 70% Ethanol for long-term storage
b. 95% EtOH (30 min)
c. 100% EtOH (30-60 min)
d. Xylene (30 min under a fume hood)
e. Xylene (30 min under a fume hood)
f. Rinse tissues in Paraffin (melting temp of wax, 56 oC for paraplast X-tra)
g. Paraffin (melting temp of wax, 56 oC for paraplast X-tra)

*NOTE: Steps 3a to 3e can be done in bulk using plastic histology containers placed on a shaker. In steps 3f to 3g transfer the tissue to a 1.5 ml labeled eppendorf tube and add melted Paraffin → place in a 56 oC heated water bath and floating tube rack.

4. Embedding
a. Spray the stainless steel cassette with ‘base mold release agent’ prior to adding heated wax.
b. Pour a small amount (1/3 of the volume) of heated wax into a pre-heated stainless steel cassette and arrange tissue pressing it flat against the bottom with a pre-heated instrument.
c. Place the back side of the plastic labeled cassette on top of the stainless steel cassette and fill until wax just covers mesh.
d. Chill until paraffin is completely solidified and then remove stainless steel cassette.

Sectioning
1) Section 10-15 micron sections from cold blocks of paraffin imbedded tissue with cold blade (pre-chilled in 4 oC).
2) Pretreatment of paraffin tissue sections with either of the following
a. Leave slides at room temperature for 60 minutes; or
b. Heat in dry oven at 55°C for 20 minutes
Note 1: for adipose, leaving at room temperature prevents disruption of the tissue
Note 2: For RNA and antigens option (b) is recommended. Option (a) is for highly heat sensitive molecules.
3) Immerse slide in xylene for 2 min. (fat) or 10 min. (liver).
4) Immerse slide in 100% ethanol for 2 minutes.
5) Immerse slide in 95% ethanol for 1 minute.
6) Immerse slide in 70% ethanol for 1 minute.
7) Immerse slide in 50% ethanol for 1 minute.
8) Immerse slide in 1X PBS for 2 minutes.
9) Air dry slides for 10-20 min

Protocol Edited from: several online IHC protocols

H&E Staining

Hematoxylin staining
Incubate slides in Mayer’s Hematoxylin for 20-30 min (fat) or 10 min (liver)
Rinse in warm H2O (note ‘blueing’ of nuclei)

Eosin staining
Working solution:
• 25 mL of 1% Eosin (dissolved in 2 parts of ddH2O and 8 parts of 95% Ethanol)
• 75 mL of 80% Ethanol
• 0.5 mL Glacial Acetic Acid

Stain sections for 10 min. in 0.25% Eosin solution
Rinse slides in ddH2O

Dehydrate slides in
1) Immerse slide in 70% ethanol (4 dips)
2) Immerse slide in 95% ethanol (2 dips).
3) Immerse slide in 100% ethanol (2 dips).
4) Immerse slide in xylene for (6 dips)

Mounting
Apply a drop of mounting medium over the section
Place the cover slip over in an angle to avoid bubbles
Store slides at room temperature for 2 h prior to analysis

Differentiation of 3T3-L1 Cells

2013-12-18T07:59:17Z

Nporitsa:

__NOTOC__

==Materials==
*'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
*'''FBS''' (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
*'''NCS''' (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
*'''PSG''' (100X Invitrogen Cat# 100378-016)
*'''Insulin''' (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
*'''Dexamethasone''' (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
*'''MIX''' (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
*'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
*'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
*'''DMI Media''' (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
*'''Insulin Media''' (Adipocyte Media + Insulin (1000X dilution); Sterile filter into new bottle after adding insulin)

[[File:3T3-L1_Adipocyte_Differentiation_Schematic.png]]

==Fibroblast Culture==
*Cells can be grown at 37C + 8% CO2, and split 10-20X. Typically healthy fibroblasts will repopulate in 2-3 days after a 10X dilution.
*Split cells when at about 80% confluence. Confluence initiates the differentiation program, so if cells reach confluence, you will need to thaw another DMSO stock and start over. Splitting cells will normally not reverse this
*When splitting cells, wash cells 2x with sterile PBS (-/-) then add 0.05% trypsin and sit at room temperature for 2-5 min. Be careful not to over-trypsinize cells
*Cells can normally be passaged up to about passage # 25 without problems.
*Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes)

==Differentiation of Fibroblasts to Adipocytes==
*Let cells grow 2-3 days after confluence (typically 8-10 days after a 10X passage)
*Add Differentiation Media I and incubate 3-4 days. Cells should shrink after day 1 then start to round up and by *day 3 should begin to accumulate lipid (the media should change color as well)
*After 3-4 days, add Differentiation Media II and incubate 2-3 days (for a total of 6 days).
*Change media to Adipocyte media and change every 2-3 days. Insulin responsiveness (as measured by glucose uptake) increases to a maximum at about 10 days post-differentiation.
*Good cells should be homogeneous and have >10 fold insulin stimulated glucose uptake.
*If adipocytes need to be trypsinized for replating or electroporation this is best done before the fourth day after changing to Adipocyte media. Use 0.25% trypsin. After that, cells do not trypsinize well.

==Reference==
PMID 16472699
[[ Category:Cell Culture ]]

Protocol: Adipogenic Differentiation of Mouse Embryonic Fibroblasts (MEFs)
Strains: Wild type (Tsc2 +/+) and Tsc2 -/- (p53−/−) MEFs
Source: DJ Kwiatkowski (Brigham and Women's Hospital, Boston, MA)
Papers:
• Kwiatkowski DJ, Zhang H, Bandura JL, Heiberger KM, Glogauer M, et al. (2002) A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in TSC1 null cells. Hum Mol Genet 11: 525–534.
• Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, et al. (2003) Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 112: 1223–1233.

Materials
• DMEM (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)
• FBS (we test several batches from several sources for differentiation and insulin stimulated glucose uptake then order large quantities of that particular lot. Currently we are using Sigma cat# F2442, lot# 072K8403. Aliquot and store in 50 mL tubes at -20)
• NCS (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store at -20)
• Penicillin/Streptomycin (100X Invitrogen Cat# 100378-016)
• Insulin (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid (28.7μl/50ml) as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20
• Dexamethasone (Sigma D-1756). Dissolve in ethanol as a 2.5 mM stock (0.049g/50ml). Aliquot into 1.5 tubes and store at -20.
• MIX (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL. Aliquot into 1.5 mL tubes and store at -20
• Fibroblast Media (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
• Adipocyte Media (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)
• DMI Media (Adipocyte Media + Insulin (1000X dilution), Dexamethasone (10 000X dilution) and MIX (500X dilution); Sterile filter into new bottle after additions
• Insulin Media (Adipocyte Media + Insulin; Sterile filter into new bottle after adding insulin)

Induction of MEF differentiation:
1. Seed MEFs at 100% confluence in 12-well plates and grow for 2 days (D0).
2. On day 0 (D0) treat MEFs with differentiation-inducing media containing insulin (830 nM), dexamethasone (1 µM), and 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM).
3. At 48 hours, replace the media with the maintenance DMEM medium containing 10% FBS and insulin (830-nM), followed by a replenishment of the media every 2 days until the end of adipogenic differentiation (prior to cells lifting off).
• Note1 Cells should shrink after day 1, and then start to round up. By *day 3 cells should begin to accumulate lipid (the media should change color as well)
4. Protein lysate and RNA extraction is taken at day 4 (D3).
• Note2: In the 12-well plate, Tsc2-/- MEFs started to be detached from the plate on D3, and consequently lysates were taken at that point to ensure adequate sample collection.
• Note3: A 6-well plate can also be used to increase surface area and protein/RNA content from each treatment. Seeding density of 90% followed by 2 days of growth prior to adipogenic differentiation may increase adherence and maintenance of Tsc2-/- MEFs.