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08-ExpProtocols.Rmd
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08-ExpProtocols.Rmd
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# Experimental Protocols{#protocols}
## Metabolomics sample extraction{#extraction}
### Cell culture experiments
#### Cell extracts in MeOH{#ccextraction_meoh}
Materials:
* Chloroform
* 15 ml falcon tubes
* thermo or rotary shaker
Procedure:
* Add 1 ml chloroform to 5 ml methanolic cell extract
* Incube for 60 min at 4°C on rotary or thermo shaker
* Centrifuge at max speed for 10 min at 4°C
* Collect polar and lipid phases into fresh falcons / tubes
* Dry under vacuum
In order to generate technical backups:
* Resuspend dried extracts in 600 ul 20\% MeOH
* Shake at 4°C on thermo shaker for 30 min
* Split volumes into equal parts in fresh eppendorf tubes
* Dry under vacuum
Suggested cell density: $2-3e+6$ cells / extract.
#### Cell pellets{#ccextraction_susp}
Materials:
* [MCW suppl. cinnamic acid](#mcw)
* 15 ml falcon tubes
* thermo or rotary shaker
Procedure:
* Resuspend cell pellets in 1 ml MCW
* Incube for 60 min at 4°C on rotary or thermo shaker
* Add 0.5 ml of H2O to induce phase separation
* Centrifuge at max speed for 10 min, cold temperatures
* Collect polar and lipid phases into fresh falcons / tubes
* Dry under vacuum
In order to generate technical backups:
* Resuspend dried extracts in 600 ul 20\% MeOH
* Shake at cold temperature on thermo shaker for 30 min
* Split volumes into equal parts in fresh eppendorf tubes
* Dry under vacuum
Suggested cell density: $2-3e+6$ cells / extract.
### Tissue samples
Materials:
* [MCW suppl. cinnamic acid](#mcw)
* ddH20
* Eppendorf tubes
* Tissue lyzer / pulverizer
Procedure:
* Snap-freeze tissue samples
* Pulverize samples
* Aliquote 50 mg of tissue powder
* Add 1.5 ml of MCW (suppl. with cinnamic acid final conc. 2 ug/ul)
* Shake for 60 min on rotary shaker at 4°C
* Add 0.5 ml ddH20 for phase separation
* Centrifuge maximum speed, 10 min at 4°C
* Collect polar and lipid phases in fresh vessels
* Dry under vacuum
### Blood samples
Material:
* Minivette for capillary blood samples
* [MCW suppl. cinnamic acid](#mcw)
* ddH20
* Eppendorf tubes
Procedure:
* Give 20 ul blood / sera directly into 1 ml MCW to avoid lumps
* In case of lumps sonicate samples
* Shake samples at 4°C for 800 rpm for 60 min
* Add 500 ul ddH20 and vortex shortly
* Spin down at 4°C at max speed for 10 min
* Aliquote polar phase into 2-3 times 500 ul in 1.5 ml tubes
* Aliquote lipid phase 2 times in 100 ul lower in 1.5 ml eppi
* Dry in SpeedVac (35°C)
## Sample derivatisation
### Standard protocol
Materials:
* Methoxamine (MeOx)
* Pyridine (open under the hood only!)
* MSTFA
* [Alkane mix](#alkanemix) (c10-c36) in Hexane
* Chromacol vials and caps (big, small)
Mixtures:
* Solvent 1: 40 mg MeOx in 1 ml Pyridine
* Solvent 2: 10 ul Alkane mix in 1 ml MSTFA
Volumens of both solvents are shown for standard (small volumes) procedures.
Procedure:
* Make sure samples are completly dry (1 h speed vac)
* Add 20 ul (10 ul) of solvent 1 / sample
* Incubate on rotary shaker, 30°C, for 60 min
* Add 80 ul (25 ul) of solvent 2 / sample
* Incubaate on rotary shaker, 37°C, for 90 min
* Centrifuge to spin down insoluble materials
* Prepare aliquotes three times 28 ul or two times 15 ul (small glass vials)
* Keep on room temperature until measurement (max. 10 days)
### Specific protocols
#### Andrash
#### Fabian
#### Henning