Technical information

Laura Corns; Andrew Metcalfe; and Elena Rainero

Preparation of drugs

For session 1, only DMSO and NSC23766 need to be prepared and available to students.

For session 2, all drugs should be prepared and made available to students. To help prevent excess drugs being prepared, a Google form (or similar) could be prepared which students complete in advance of the session to allow appropriate preparation.

 

Drugs Target Stock to prepare Working concentration Safety
DMSO Negative control 10% in 100 µl aliquots 0.01% Flammable
NSC23766 Rac1 inhibitor – positive control 1 mM in water in 200 µl aliquots 25 µM
EGF (epidermal growth factor) Upregulates NaV expression 1 mg/ml in water in 100 µl aliquots 30ng/ml Non
PF-04531083 Selective NaV1.8 inhibitor 100 µM in 10% DMSO in 100 µl aliquots 1 µM (max) H302 Harmful if swallowed.
PF-05089771 Selective NaV1.7 and NaV1.8 inhibitor 100 µM in 10% DMSO in 100 µl aliquots 1 µM (max) Possible irritation of throat, skin and eyes if contact made

Preparation of A2780-Rab25 cells

To prepare the 6 well plates of A2780-Rab25 before sessions 1 and 3:

  1. Prepare an adequate volume of growth media: RPMI-1640 medium + 10% Fetal Bovine Serum + 1% PenStrep (10,000 U Penicillin and 10 mg/ml Streptomycin).
  2. Complete the following protocol using a class II microbial safety cabinet and sterile technique to avoid contamination of the cell culture.
  • Discard existing media and wash with 1x PBS (2.5 ml for T12.5, 5 ml for T25, 10 ml for T75).
  • Add 0.25% Trypsin-EDTA (300 µl for T25, 700µl for T75) and incubate for 3 min at 37℃. Check cells have detached using a microscope and tap the flask if required.
  • Add growth media to deactivate the Trypsin (4.7 ml for T25, 9.3 ml for T75). If necessary, gently pipette the media down the side of the flask to detach cells from the flask surface.
  • Seed 6 well plates, with approximately 20,000 cells, in a total volume of 2 ml per well.
  • Incubate for 3 days at 37℃ 5% CO2 for 3 days (for example, seed Monday for use on Thursday). It is not necessary to change the media during this time as the students will change the media during the session.

An incubator set at 37℃, 5% CO2 must be left available on the day between session 1 and 2, and between session 3 and 4 to allow incubation of the cells and encourage migration.

Equipment

Each group of 4 should have a bench with the following:

  • 2 x 6 well plates with confluent monolayers of A2780-Rab25 cells
  • Fine marker pen
  • p10 pipette and tips
  • p200 pipette and tips
  • p1000 pipette and tips
  • pipettor (also known as a PIPETBOY)
  • plastic serological pipettes (which are used with the pipettor)
  • sterile media
  • 1 x eppendorf of DMSO (10%)
  • All of the VGSC inhibitor stock solutions available in session 3 will be dissolved in DMSO. This is a solvent that is known to directly effect some cell effects. Following dilution of the VGSC inhibitors from stock to working concentrations, the maximum concentration of DMSO that cells would be exposed to is 0.1% DMSO
  • 1 x eppendorf of 1mM NSC23766
  • NSC23766 inhibits Rac1 at a concentration of 25μM. Rac1 is a small GTPase that is essential for cell migration.
  • Inverted microscope with phase contrast. EVOS Core XL were used

Licence

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Cat burglars, yeast races, and other hypothesis-driven bioscience practicals Copyright © 2024 by The authors and the University of Sheffield is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.

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