Differentiation of 3T3-L1 Cells - Revision history

Reddj: added SOP requirements

2020-06-04T21:56:59Z

added SOP requirements

← Older revision		Revision as of 21:56, 4 June 2020
Line 1:		Line 1:
	__NOTOC__		__NOTOC__
			==SOP==
			*[[SOP- Biosafety Cabinets]]
			*[[Sop- Compressed Gases]]
			*[[SOP- Vacuum Pumps]]

	==Materials==		==Materials==

Davebrid: /* Materials */ updated concentration of IBMX

2018-03-18T15:04:55Z

Materials: updated concentration of IBMX

← Older revision		Revision as of 15:04, 18 March 2018
Line 8:		Line 8:
	*'''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		*'''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.		*'''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		*'''MIX''' (Sigma I-5879). Add 2.78g MIX and 0.98g KOH and bring up to 50 mL (250 mM stock). Aliquot into 1.5 mL tubes and store at -20
	*'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)		*'''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)		*'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)

Nporitsa: /* Reference */

2013-12-23T19:34:37Z

Reference

← Older revision		Revision as of 19:34, 23 December 2013
Line 34:		Line 34:
	PMID 16472699		PMID 16472699
	[[ Category:Cell Culture ]]		[[ 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.

Nporitsa at 07:59, 18 December 2013

2013-12-18T07:59:17Z

← Older revision		Revision as of 07:59, 18 December 2013
Line 34:		Line 34:
	PMID 16472699		PMID 16472699
	[[ Category:Cell Culture ]]		[[ 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.

Davebrid: updated MIX stock solution instructions

2013-09-23T14:17:35Z

updated MIX stock solution instructions

← Older revision		Revision as of 14:17, 23 September 2013
Line 8:		Line 8:
	*'''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		*'''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.		*'''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). ~~Dissolve~~ 2.78g~~/50mL KOH (~~0.98g~~/50mL)~~. Aliquot into 1.5 mL 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)		*'''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)		*'''Adipocyte Media''' (DMEM + 10 % L1-FBS + 1X PSG; Sterile filter into new bottle)

Lubinbin: /* Fibroblast Culture */

2009-10-26T17:26:56Z

Fibroblast Culture

← Older revision		Revision as of 17:26, 26 October 2009
Line 19:		Line 19:
	*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.		*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		*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		*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.		*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)		*Plate cells out in whichever format you require your adipocytes in (i.e. 12 well plates or 15 cm dishes)

Lubinbin: /* Fibroblast Culture */

2009-10-26T17:25:06Z

Fibroblast Culture

← Older revision		Revision as of 17:25, 26 October 2009
Line 17:		Line 17:

	==Fibroblast Culture==		==Fibroblast Culture==
	*Cells can be grown at 37C + 5% CO2, and split 10-20X. Typically healthy fibroblasts will repopulate in 2-3 days after a 10X dilution.		*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		*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		*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

Lubinbin: /* Materials */

2009-10-26T17:24:03Z

Materials

← Older revision		Revision as of 17:24, 26 October 2009
Line 6:		Line 6:
	*'''NCS''' (currently we are using Biowhittaker Cambrex cat# 14-416F, lot# 01106776; aliquot in 50 mL tubes and store 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)		*'''PSG''' (100X Invitrogen Cat# 100378-016)
	*'''Insulin''' (Sigma I-5523). Dissolve in 0.01N Hydrochloric acid as a 1 mg/ml stock. Aliquot into 1.5 mL tubes and store at -20		*'''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. Aliquot into 1.5 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). Dissolve 2.78g/50mL KOH (0.98g/50mL). Aliquot into 1.5 mL tubes and store at -20		*'''MIX''' (Sigma I-5879). Dissolve 2.78g/50mL KOH (0.98g/50mL). Aliquot into 1.5 mL tubes and store at -20
	*'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)		*'''Fibroblast Media''' (DMEM + 10% NCS + 1X PSG; Sterile filter into new bottle)
	*'''Adipocyte Media''' (DMEM + 10 % FBS + 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		*'''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)		*'''Insulin Media''' (Adipocyte Media + Insulin (1000X dilution); Sterile filter into new bottle after adding insulin)

Davebrid at 14:50, 20 October 2009

2009-10-20T14:50:04Z

← Older revision		Revision as of 14:50, 20 October 2009
Line 21:		Line 21:
	*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		*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.		*Cells can normally be passaged up to about passage # 25 without problems.
	*Plate cells out in whichever format you require your adipocytes in (ie 12 well plates or 15 cm dishes)		*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==		==Differentiation of Fibroblasts to Adipocytes==

Davebrid at 13:34, 27 July 2009

2009-07-27T13:34:09Z

← Older revision		Revision as of 13:34, 27 July 2009
Line 1:		Line 1:
			__NOTOC__

	==Materials==		==Materials==
	*'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)		*'''DMEM''' (4.5 g/L glucose + L-glutamine – sodium pyruvate; Invitrogen catalog # 11965-092)