Introduction

The procedure presented below describes a facile method for studying signal transduction events with adherent cells (HeLa, MCF-7, BALB/c 3T3, etc.) and suspension cells (Jurkat, Raji, THP-1, etc.). In this protocol, suspension cells are plated into the wells of a 96-well microplate while adherent cells are detached from their culture vessel using a dissociation reagent, then placed into wells of a 96-well microplate. Cells are stimulated as desired. At the end of stimulation, cell culture medium is removed from the bottom of the wells by gentle aspiration using a vacuum manifold. The cells are then washed with PBS, aspirated, and lysed within the wells by the addition of cell extraction buffer. The cell extracts are then assayed using Invitrogen phosphoELISA kits.

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Adherent cells preparation

Materials

  • 96-well microplates (Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate)
  • T-75 cell culture flask (Nunc EasYFlask Cell Culture Flasks)
  • Conical tubes (Nunc 15mL Conical Sterile Polypropylene Centrifuge Tubes)
  • Complete cell culture medium (e.g., DMEM plus 10% FBS)
  • Trypsin-EDTA solution (Trypsin-EDTA (0.05%), phenol red) (See other cell dissociation reagents)
  • Phosphate-buffered saline (PBS (10X), pH 7.4)
  • Orbital plate shaker
  • Vacuum manifold
  • Protease inhibitors (Halt Protease Inhibitor Cocktail (100X))
  • Cell extraction buffer (10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate)

    • Note: 1X Cell extraction buffer may be divided into 1X aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting the cells.

    Additional recommended materials

    Protocol tips

    Add protease inhibitor directly to the cell extraction buffer or the extract. We recommend using Halt Protease InhibitorCocktail, reconstituted to 1X (10 μL of 100X Halt Protease Inhibitor Cocktail per mL of cell extraction buffer). The stability of protease inhibitor-supplemented cell extraction buffer is 24 hours at 4°C.


    Protocol

    1. Grow cells to desired level of confluency in a T-75 flask.
    1. Decant or aspirate the medium.
    1. Add 2–3 mL fresh warm Trypsin/EDTA solution. Transfer the flask to a 37°C incubator.

    Note: There are other cell dissociation products and enhanced cell trypsinization solutions that are available. Please refer to Cell Dissociation and Trypsin for Cell Culture.

    1. Wash with warm PBS. Aspirate.
    1. After 5 minutes, tap the side of the flask, and examine the flask under a microscope for lifting. If necessary, return the cells to the incubator for an additional 5–10 minutes, with occasional tapping, until lifting is complete.
    1. Quickly quench the trypsin reaction by adding 5–6 mL of complete cell culture medium.
    1. Transfer the cells to sterile 15 mL conical tubes.
    1. Pellet the cells by centrifugation at 300 x g for 7 minutes.
    1. Decant the supernatant.
    1. Wash the cells by pipetting 10 mL medium into each conical tube and resuspending the pellet. Collect the cells by centrifugation at 300 x g for 7 minutes.
    1. Resuspend the washed cells in complete cell culture medium.
    1. Enumerate cell density. For most applications, the cell density should be adjusted to 5–25 x 104 cells/mL cell culture medium. It is important to note that this value may require some optimization for each specific application. Cell doubling time is an important factor to be considered when adjusting cell density at the beginning of an experiment.
    1. Plate 200 µL of cell culture (i.e., 10,000–50,000 cells) into the wells of a sterile 96-well cell culture plate. Incubate the cells for 18 hours at 37°C. The filter plate is designed to retain the particles, while permitting the flow of liquids from the bottom of the plate.
    1. Stimulate the cells as desired. For example, HeLa cells can be stimulated with 100 μM of anisomycin for 60 minutes at 37°C.
    1. At the end of the stimulation, place the 96-well plate on the vacuum manifold and remove cell culture medium from the bottom of wells by gentle aspiration.
    1. Wash the cells by pipetting 200 µL of ice-cold PBS into each well. Remove the PBS from the bottom of the wells by gentle aspiration. Repeat the wash step two times for a total of three washings.
    1. Pipette 25 µL of protease inhibitor-supplemented Cell extraction buffer into each well. Incubate the plate on ice for 30 minutes.
    1. Thoroughly mix the contents of each well by pipetting up and down 5–6 times. A multi-channel pipette is desirable for this application. This causes the cells to lyse. At this point in the procedure, the extracts are ready for analysis. Alternatively, the extracts may be stored in the plate at –20°C for future analysis. Frozen plates should be thawed on ice in preparation of completing the assays.
    1. Place the plate on an orbital shaker and mix for 1 minute.
    1. Prepare the phosphoELISA kits. Sample wells: Pipette 95 µL of Standard Diluent Buffer (included in the kits) into the wells of the phosphoELISA plates designated for samples. Transfer 5 µL of cell extract from the filter plate into the sample wells of the plates. Place the plates on an orbital shaker to thoroughly mix the contents of the wells. Standard wells: Prepare standards as indicated in the assay protocol and pipette into the designated wells.
    1. Complete the phosphoELISA as directed by the assay protocol.


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    Suspension cells preparation

    Materials

    Note: 1X Cell extraction buffer may be divided into 1X aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting the cells.

    Additional recommended materials

    Protocol tips

    Add protease inhibitor directly to the cell extraction buffer or the extract. We recommend using Halt Protease InhibitorCocktail, reconstituted to 1X (10 μL of 100X Halt Protease Inhibitor Cocktail per mL of cell extraction buffer). The stability of protease inhibitor-supplemented cell extraction buffer is 24 hours at 4°C.


    Protocol

    1. Grow cells to desired level of confluency in a T-75 flask.
    1. Decant or aspirate the medium.
    1. Transfer the cells to sterile 15 mL conical tubes.
    1. Pellet the cells by centrifugation at 300 x g for 7 minutes.
    1. Decant the supernatant.
    1. Wash the cells by pipetting 10 mL medium into each conical tube and resuspending the pellet. Collect the cells by centrifugation at 300 x g for 7 minutes.
    1. Resuspend the washed cells in complete cell culture medium.
    1. Enumerate cell density. For most applications, the cell density should be adjusted to 25–100 x 104 cells/mL cell culture medium. It is important to note that this value may require some optimization for each specific application. Cell doubling time is an important factor to be considered when adjusting cell density at the beginning of an experiment.
    1. Plate 200 µL of cell culture (i.e., 50,000–200,000 cells) into the wells of a sterile 96-well filter bottom plate. Incubate the cells for 24 hours at 37°C. The filter plate is designed to retain the particles, while permitting the flow of liquids from the bottom of the plate. 
    1. Stimulate the cells as desired. For example, Jurkat cells can be stimulated with 20 μg/mL of anisomycin for 60 minutes at 37°C.
    1. At the end of the stimulation, place the 96-well plate on the vacuum manifold and remove cell culture medium from the bottom of wells by gentle aspiration.
    1. Wash the cells by pipetting 200 µL of ice-cold PBS into each well. Remove the PBS from the bottom of the wells by gentle aspiration. Repeat the wash step two times for a total of three washings.
    1. Pipette 25 µL of protease inhibitor-supplemented Cell extraction buffer into each well. Incubate the plate on ice for 30 minutes
    1. Thoroughly mix the contents of each well by pipetting up and down 5–6 times. A multi-channel pipette is desirable for this application. This causes the cells to lyse. At this point in the procedure, the extracts are ready for analysis. Alternatively, the extracts may be stored in the plate at –20°C for future analysis. Frozen plates should be thawed on ice in preparation of completing the assays.
    1. Place the plate on an orbital shaker and mix for 1 minute.
    1. Prepare the phosphoELISA kits. Sample Wells: Pipette 95 µL of Standard Diluent Buffer (included in the kits) into the wells of the phosphoELISA plates designated for samples. Transfer 5 µL of cell extract from the filter plate into the sample wells of the plates. Place the plates on an orbital shaker to thoroughly mix the contents of the wells. Standard Wells: Prepare standards as indicated in the assay protocol and pipette into the designated wells.
    1. Complete the phosphoELISA as directed by the assay protocol.


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    Stylesheet for Classic Wide Template adjustments

    For Research Use Only. Not for use in diagnostic procedures.