Jan 11, 2022

Public workspaceIntegrated Extraction for Cells

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3p5bql25/v1
  • Juan Sanchez1
  • 1CZ Biohub
  • Juan Sanchez: Stanford
Juan Sanchez
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3p5bql25/v1
Protocol Citation: Juan Sanchez 2022. Integrated Extraction for Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p5bql25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 11, 2022
Last Modified: January 11, 2022
Protocol Integer ID: 56768
Keywords: liquid extraction for cell, integrated extraction for cell, liquid extraction, metabolomics sample, metabolomics samples for hilic, thesimultaneous generation of lipidomic, integrated extraction, lipidomic, c18 chromatography, liquid, cell
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Abstract
This liquid-liquid extraction for cells enables thesimultaneous generation of lipidomics and metabolomics samples for HILIC and C18 chromatography.
Guidelines
This extraction should be performed in a fume hood.
Materials
Equipment

  • Qiagen Bead Beater
  • Centrifuge
  • Mixmate Shaker
  • Sonicator
  • Centrifugal evaporator
  • P200 Micropipette
  • P1000 Micropipette
  • P200 Micropipette tips
  • P1000 Micropipette tips

Consumables

  • Stainless steel balls 2.3 mm
  • 96-well plate polypropylene
  • 1.5ml amber glass vial
  • 150 ul glass vial inserts
  • 2ml round bottom Eppendorf polypropylene tube with cap

Chemicals

  • Water
  • Acetonitrile
  • Methanol
  • MTBE
  • HILIC iSTDs




Troubleshooting
Cells should arrive pelleted and frozen from collaborator
Using wide-mouth pipette wash pellet with 500uL 150 mM ammonium acetate (25x aspirations)
Centrifuge @ 200 g for 5 min
Remove supernatant
Repeat steps 2-4 a second time
Add 4-6 non-sterile 2.3mm stainless steel beads to the tube.
Dispense 225 ul of ice-cold Methanol at -20 deg C containing 1.5% iSTD “SPLASH” mix into extraction tube 1.
Seal the sample tube and place it in the bead beater at an amplitude 20 Hz for 15 minutes to homogenize the sample & extract the metabolites.
Close the tube.
Vortex extraction tube 1 for 10 seconds.
Open extraction tube 1 and dispense 750ul of ice-cold MTBE.
Close extraction tube 1 and vortex for 10s.
Shake extraction tube 1 for 6 min at 4 deg C.
Open extraction tube 1 and dispense 188 ul of LC-MS grade water containing the 18 HILIC iSTDs at 1/3rd of their normal concentration as defined in the HILIC extraction SOP except CUDA.
Close extraction tube 1 and vortex it for 20 seconds
Centrifuge extraction tube 1 for 2 min at 14,000xg.
Open extraction tube 1 and transfer 700ul of the upper phase into a single 700ul tubetwo new separate tubes(350ul/ tube); henceforth referred to as (Lipidomics tube 1) and (Lipidomics tube 2).
Transfer 250ul of the bottom phase from extraction tube 1 into a new single-tube hereafter referred to as (Hilic tube 1).
Centrivap Lipidomics tube 1, and Hilic tube 1 at room temperature until dry.
Reconstitute Lipidomics tube 1 in 120 ul of Methanol containing CUDA istd and close the tube.
Perform lipid cleanup on HILIC tube 1: Add 500uL of 1:1 ACN:Water and vortex for 20s
Centrifuge at 14,000 g for 2 min.
Transfer supernatant to a new tube and dry down
Reconstitute HILIC tube 1 in 60ul of ACN:H2O (4:1 v/v) containing CUDA and close the tube.
Sonicate Lipidomics tube 1 and Hilic tube 1 at room temperature for 5 min.
Centrifuge Lipidomics tube 1 and Hilic tube 1 at 14,000xg for 2 min.
Open lipidomics tube 1, transfer equal fractions of the extract into two separate amber glass vials with micro-inserts, hereafter referred to as (lipidomics vial 1 pos) and (lipidomics vial 1 neg), and close the vials.a
Open Hilic tube 1 and transfer the extract into an amber glass vial with a micro-insert, hereafter referred to as (Hilic vial) and close the vial.
Analyze the samples via UHPLC–Orbitrap as follows.