Metabolite extraction

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Contents

Protocol 1.1 (Staphylococcus_Filtration)

Intro: An optical density (O.D.) 600 of 10 was achieved for the total number of cells collected for one dimensional (1D) 1H NMR, or an O.D. 600 of 20 was collected for two dimensional (2D) 1H-13C HSQC NMR experiment. To reach targeted O.D. 600, 14-16 mL of culture was used for each sample for three hour growth and 3-5 mL was used for eight hour growth.

Steps:

1. Millipore® Microfil ® V filtration system (0.45 micrometer pore size, pre-sterilized filter paper) was used to separate cells from the growth media. Each filter paper was pre-washed immediately before use, with 10 mL 20 mM phosphate buffer (pH 7.2) and was used for collecting one culture sample.

2. After the filtration of bacteria cells, the filter membranes were placed into 50 mL Falcon tubes (pre-cooled at -70oC).

3. To quench the cells, the tubes were sealed, and put into a bucket of liquid nitrogen immediately.

4. The cells were transferred to a 1.5 mL tube (pre-cooled at -20°C) from each filter membraneindividually by suspension with 1 mL ice-cold 20 mM phosphate buffer.

5. The cells were spun down at 13200 rpm for one minute at -9°C to remove the residue media, and were re-suspended in 1 mL ice-cold phosphate buffer.

6. The O.D. 600 (1:100 - 1:200 dilution) of the cells from each sample was measured and adjusted to a targeted O.D. 600 in 1 mL phosphate buffer.

7. FastPrep® system (MP Biomedicals) was used to lyse the cells. The cells from each sample were disrupted by glass bead in a 2 mL tube (pre-cooled at -20oC) twice following a pre-set cycle (Speed 6, 40 s, Program 1) with a 5 minute rest time.

8. The tubes were centrifuged at 13200 rpm for two minutes at -9°C.

9. 700 µL supernatantin each tube was transferred into another 2 mL fresh tube (pre-cooled at -20°C).

10. An extra washing step for the cell debris with 1 mL ice-cold phosphate buffer was performed and 900 µL of supernatant was taken and pooled with the previous 700 µL extraction.

11. A final 1.5 mL sample in each fresh tube was prepared without any glass beads.

12. All tubes were flash frozen in liquid nitrogen and then stored in dry ice for a short transportation period.

13. The samples were lyophilized completely. 600 µL D2O was added to the lyophilized samples. 50 µM TMSP for 1D 1H NMR or 500 µM TMSP for 2D 1H-13C HSQC NMR was also added to each sample as an internal standard. The samples were held in 5 mm NMR tubes (Norell ST500-7, Norell, Inc., Landisville, NJ USA).

Protocol 1.2 (Staphylococcus_Centrifugation)

1. Streak the plates for individual colonies.

2. Pick up one colony for each test tube of 3 mL TSB media and inoculate it for a day (~9 hours). Do necessary repeats with different cultures.

3. Titrate 15 µL pre-culture being grown during the day into 3mL TSB for overnight 12 hour growth for each culture.(1:200 ratio dilution).

4. Use UV/Vis spectrometer to find out O.D. Dilute the sample into 1:20 when necessary to keep the O.D. value in its effective range of reading.

5. For 2h growth, in order to have enough cells, 36 mL TSB media is used and for 6h growth, 18 mL TSB media is used. We have two types of flasks: 125 mL and 250 mL. So 18 mL media is kept in the smaller flask and 36 mL media will be held in the large flask. In both cases, the ratio of the media and the total would be 1:7 so that we will have enough oxygen during the growth.

O.D. and pH after growth are recorded.

6. All growths are in constant 37oC.

7. Lysate:40ms, program 1 Spin down: 5 min maximum speed (132000 rpm)

8. Centrifuge: rotor->29->SH-3000BK->4000->12 min

NOTE: KEEP 500uL of MEDIA FOR NMR use each time

Protocol 2 (Mycobacteria_Centrifugation)

1. All bacterial culture were grown in 50mL of Middlebrook 7H9 media for roughly 14 hours at 37oC with shaking at 200 rpm until an OD600=0.6 was achieved.

2. The cultures were placed on ice for 5 minutes, and centrifuged for 10 minutes at 2,700 rpm.

3. The used media was removed and the cell pellets were washed three times with 30 ml of ice cold double distilled water.

4.The washed cell pellets were resuspended with 10 ml of double distilled water and transferred to 30mL Pyrex beakers.

5. The cell pellets were then sonicated on a salt-ice water bath with a Vibra Cell Model VC600 for 5 minutes in the presence of 30% (vol/vol) type A-5 alumina.

6. The cells were centrifuged for 30 minutes at 15,000 rpm, and the supernatant was collected to remove any cell debris.

7. The supernatant was transferred to a 50ml Corning tubes and frozen in an ethanol-dry ice bath and stored at -80oC until ready to be analyzed.

8. The supernatant was lyophilized and resuspended with 700 uL of 99.8% D2O containing 50 mM phosphate buffer (pH=7.2) with 50 uM of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP).

9. The samples were vortexed and centrifuged for 3 minutes at 13,000 rpm, and 600 uL of the cell free extract was transferred to NMR tubes.

NOTE: KEEP 500uL of MEDIA FOR NMR use each time


Protocol 3 (Pancreatic cancer and Neuroblastoma cells_MeOH extraction )

1. Aspirate off medium.

NOTE: KEEP 500uL of MEDIA FOR NMR use each time

2. Tilt plate, aspirate of all media.

3. Keeping plated tilted, wait a few seconds to allow any additional media to collect in corner of plate, and them aspirate to get off as much as possible.

4. Add 5 mL of phosphate buffer and wash twice.

5. Immediately add 1 mL of 80% methanol (-80°C).

6. Place the plates at -80 °C for 15 minutes.

7. Scrap plates on dry ice with cell scraper.

8. Transfer cell lysate/methanol mixture to 2.5 mL eppendorf tube.

9. Centrifuge at full speed for 5 minute in cold room to pellet cell debris and proteins.

10. Transfer the supernatant to 2.50 mL eppendorf tube.

11. Re-extract the cell pellet with 0.25 mL of water.

12. Add to the sup collected from 80% methanol extract.

Protocol 4 (Parkinson disease cells_MeOH extraction )

Protocol 5(Biotissue_polar)

Extraction of polar metabolites from tissues using methanol

1. Prepare ice cold 80% methanol

2. Sterilize the mortar and pestle by autoclaving

3. Weigh intact frozen tissue, and transfer in to a cryogenically chilled mortar.

4. Homogenize after adding 4 ml of methanol per gram of tissue

5. Leave the sample in freezer for 15 min.

6. Transfer it in to a tube

7. Centrifuge at 1,000g for 15 min at 4 oC

8. Transfer the upper phase and remove the solvent using speed vacuum concentrator

9. re-extraction can be done using water


Protocol 6 (E.Coli)

1. Prepare ice-cold 1:1 water:methanol solution to carry out the extraction

2. After the cells are in the extraction phase (Log/Stationary phase) transfer the media in to centrifuge tubes

3. Centrifuge suspension culture at 4500-5000 rpm at 4 oC for 20 min

4. Prepare an ice bath to keep cell pellets cold during extraction. Pay attention to not heat up the cell pellet during the entire process

5. Once the cells are pelleted by centrifugation. Aspirate the media using either a Falcon pipette or glass pasture pipette. If the media is useful to you store some 2-3ml/all of it in Falcon tubes. No further process is necessary if you have a clear supernatant after centrifugation

6. Add 1.5 ml of the ice-cold methanol solution to your cell pellets and using a pipette gently disperse the cell pellet

7. Sonicate the cell mixture for 5 20s bursts waiting 10 sec between each burst to avoid sample heating

8. Transfer the sonicated mixture to micro-centrifuge tubes and centrifuge the cell mixture at 13000 rpm for 5 min at 4C

9. Keep 1ml the supernatant in a new micro centrifuge tube

10. Add 0.5 ml of the ice-cold mixture and follow steps 7 and 8 and add the resulting supernatant to your tube from step 8

11. Speed vac (Rotary evaporate the methanol from the cell extract-obtained at step 10)

12. It will take a couple of hours to get rid of all methanol. At this stage snap-freeze in Liquid nitrogen (micro-centrifuge tube)

13. Set up the lyophilizer for drying the out the cell lysates and aliquote of media

14. This step should take over 8-24 hours depending on usage on the lyophilizer. After this you are ready to prepare the samples for NMR


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