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Harvesting Protocol for Adherent Cells Grown in 2D Cell Culture for Metabolomics

  • Updated July 23, 2024
This protocol explains how to harvest adherent cells grown in 2D cell culture for aqueous compounds for metabolomic assessments by Gigantest.

Table of Contents

Starting Sample Quantity

For targeted, untargeted lipidomics, metabolomics  (endogenous metabolites), drug & chemical detection: grow two sets of cells:

  • Set 1: Choose either a 10 cm dish or a 60 cm dish and ensure they reach 90-100% confluency on the day of harvest for optimal metabolite quantification.
  • Set 2: Grow an aliquot of cells in the same format to count them on the day of harvest of set 1. It’s essential to record the cell number to allow for the normalization of metabolite intensity for comparison purposes.

The bigger the sample quantity, the better the chance of detecting metabolites with lower intensities. We recommend n=3-5 (at least) for preliminary data, n > 5 for publication. If you cannot have the recommended sample quantity, please let us know so we can find the best solution.

The following protocol is for ONE sample. To collect multiple samples, use multiple tubes for each step.

Materials

  1. Make sure you have the following materials before you start harvesting:
    • HPLC-MS-grade Acetonitrile (EMD Millipore cat# AX0156-6, 1L or Thermo Scientific cat# A956-1)
    • Phosphate Buffered Saline (PBS)
    • Liquid nitrogen to snap freeze samples

Media Sample Collection From Set 1 Cells (optional)

If you want to assess metabolites in the MEDIA (in addition to assessing metabolites in the cells), follow steps 3 through 7 to collect a media sample. Otherwise, skip to step 8 to collect the cell samples.

  1. Pour the media from your dish (or flask) into a 15 mL tube
  2. Centrifuge the 15 mL tube at 1500 rpm for 10 minutes at 4˚C to separate the media from residual cells.
  3. Transfer 2 mL of the media to a new 2 mL tube
  4. Label the tubes "Media - Sample Name / Code".
  5. Snap freeze the 2 mL tube containing the media sample and store at -80˚C in a sample box.

Cell Sample Collection From Set 1 Cells

Work with ONE plate at a time and make sure to perform the following steps for each plate within 3-5 minutes.

  1. Aspirate the culture media from the dish / flask.
  2. Add 5 mL of cold PBS to the dish / flask.
  3. Aspirate out PBS with a mild vacuum.
  4. Place the dish / flask at a 30˚ angle for 1 minute on ice and aspirate remaining PBS without touching the cells. (Note: remove as much PBS as possible to minimize salt contribution)
  5. Immediately add 1000 μL of cold acetonitrile harvest solution to the dish / flask.
  6. Scrape the cells with a cell-scraper.
  7. Transfer the cells together with the solution to a 2 mL tube
  8. Label the tubes "Cells - Sample Name / Code".
  9. Snap freeze the 2 mL tube containing the pellet in liquid nitrogen and store at -80˚C in a sample box.

Packaging & Shipping

  • Place the sample boxes containing your samples ( cells and optionally media ) in a Styrofoam box full of dry ice making sure to surround the sample boxes on all sides.
  • Email the Sample Information Form to info@gigantest.com.
  • Ship via FedEx Priority Overnight or FedEx Standard Overnight, or other express carriers of your choice. Please include our phone number, (667) 312-2914, on the shipping label so that the carrier can contact us when they arrive at our location. Please also select the “Direct Signature Required” option under the “Signature options”.

Ship to:

Gigantest

31 Light Street

3rd Floor

Baltimore, MD 21202

If your samples are located in Maryland, please email Gigantest at info@gigantest.com or call (667) 312-2924 to arrange a sample pick-up. Make sure to keep the samples on dry ice.