Bridges Lab Protocols - User contributions [en]

Preparing Cell Lysates

2009-11-03T16:19:27Z

Lubinbin: /* Basic Protocol */

==Materials==

===RIPA Buffer (for 10mL lysis buffer)===
{| border="1"
! || Final Concentration || per 10 mL || Stock
|-
|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
|-
|Na Deoxycholate || 0.25% || 250uL || 10%
|-
| NP-40 || 1% || 1mL || 10%
|-
|NaCl || 150mM || 375uL || 4M
|-
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|-
|NaPPi || 25 mM || 1 mL || 250mM
|-
|Protease Inhibitors || || 1 mini tab ||
|}

==Basic Protocol==
# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
# Add 200uL Buffer (RIPA buffer) and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4oC to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-11-03T16:18:10Z

Lubinbin: /* RIPA Buffer (for 10mL lysis buffer) */

==Materials==

===RIPA Buffer (for 10mL lysis buffer)===
{| border="1"
! || Final Concentration || per 10 mL || Stock
|-
|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
|-
|Na Deoxycholate || 0.25% || 250uL || 10%
|-
| NP-40 || 1% || 1mL || 10%
|-
|NaCl || 150mM || 375uL || 4M
|-
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|-
|NaPPi || 25 mM || 1 mL || 250mM
|-
|Protease Inhibitors || || 1 mini tab ||
|}

==Basic Protocol==
# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4oC to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-11-03T16:15:59Z

Lubinbin: /* RIPA Buffer (for 10mL lysis buffer) */

==Materials==

===RIPA Buffer (for 10mL lysis buffer)===
{| border="1"
! || Final Concentration || per 10 mL || Stock
|-
|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
|-
|Na Deoxycholate || 0.25% || 250uL || 10%
|-
| NP-40 || 1% || 1mL || 10%
|-
|NaCl || 150mM || 375uL || 4M
|-
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|-
|NaPPi || 25 mM || 1 mL || 250mM
|-
|- Protease Inhibitors !! 1 mini tab
|}
|NaPPi || 25 mM || 1 mL || 250mM

==Basic Protocol==
# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4oC to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-11-03T16:14:17Z

Lubinbin: /* Basic Protocol */

==Materials==

===RIPA Buffer (for 10mL lysis buffer)===
{| border="1"
! || Final Concentration || per 10 mL || Stock
|-
|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
|-
|Na Deoxycholate || 0.25% || 250uL || 10%
|-
| NP-40 || 1% || 1mL || 10%
|-
|NaCl || 150mM || 375uL || 4M
|-
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|-
|NaPPi || 25 mM || 1 mL || 250mM
|-
|- Protease Inhibitors !! 1 mini tab
|}

==Basic Protocol==
# Stimulate cells if necessary (i.e. insulin treatment for 10 min)
# Wash cells 2x1mL (1mL per well for 12 well plates, 2mL per well for 6 well plates) with ice cold PBS -/- and aspirate
# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4oC to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and do Bradford assay before add 150uL 2XSDS
# Load gel

Preparing Cell Lysates

2009-11-03T16:10:41Z

Lubinbin: /* RIPA Buffer (for 10mL lysis buffer) */

==Materials==

===RIPA Buffer (for 10mL lysis buffer)===
{| border="1"
! || Final Concentration || per 10 mL || Stock
|-
|Tris pH7.4 || 50mM || 500uL || 1M (pH 8.0@25oC)
|-
|Na Deoxycholate || 0.25% || 250uL || 10%
|-
| NP-40 || 1% || 1mL || 10%
|-
|NaCl || 150mM || 375uL || 4M
|-
|EDTA || 1mM || 20uL || 0.5M
|-
|NaVO3 || 100uM || 10uL || 100mM
|-
|NaF || 5mM || 100uL || 0.5M
|-
|NaPPi || 25 mM || 1 mL || 250mM
|-
|- Protease Inhibitors !! 1 mini tab
|}

==Basic Protocol==
# Stimulate cells if necessary
# Wash cells 2x1mL with ice cold PBS -/- and aspirate
# Add 200uL [[Buffer:RIPA]] buffer and scrape cells
# Pipet into cold eppendorf tubes
# rotate end over end for 30 minutes at 4C to lyse
# Centrifuge 10 min at 13,000 RPM to clarify
# Transfer 150uL of lysate to fresh tube and add 150uL 2XSDS
# Load gel

Splitting Cells

2009-10-26T17:29:36Z

Lubinbin: /* Protocol */

==Materials==
*Media (L1-FBS for 3T3-L1, COS-FBS for others): Prepare by filtering together 5 mL PSG, 50 mL COS-FBS and one bottle of DMEM
*PBS -/-
*0.05% Trypsin

==Protocol==
#Warm PBS and Media in water bath
#Wash cells twice with 10 mL (per 10 cm dish) PBS -/-
#Add 1 mL trypsin and sit in the hood for 2-5 min
#Add 10 mL media to each new dish
#Check cells for trypsinization, and if necessary tap the cells
#Add 9 mL media to trypsinized cells
#Add 1 mL cells to each dish (for 10X dilution; 0.5 mL for 20X)
#Replace plates in the incubator

==Cell Specific Notes==
*3T3-L1 fibroblasts have special considerations regarding confluence. See [[Differentiation of 3T3-L1 Cells]]
*RAW 264.7 cells are scraped, not trypsinized. See [[Culturing RAW 264.7 Cells]]
*S2 cells are grown at 28C without extra CO2. See [[Culturing S2 Cells]]
[[ Category:Cell Culture ]]

Differentiation of 3T3-L1 Cells

2009-10-26T17:26:56Z

Lubinbin: /* Fibroblast Culture */

__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). 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)
*'''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 ]]

Differentiation of 3T3-L1 Cells

2009-10-26T17:25:06Z

Lubinbin: /* Fibroblast Culture */

__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). 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)
*'''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 ]]

Differentiation of 3T3-L1 Cells

2009-10-26T17:24:03Z

Lubinbin: /* Materials */

__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). 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)
*'''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 + 5% 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 ]]