Tag Archives: procedure

Fixed Cell Measurement Protocol

  1.  Put coverslip with adherent cells in the sample chamber and wash with PBS. This involves gently spraying the PBS on the coverslip with a pippette, and gently rocking the sample chamber back and forth. This should only require about 0.5 mL of PBS.
  2.  Aspirate PBS and fill chamber with 1 mL of 4% formaldehyde. Let sit for 15 minutes at room temperature (in cell prep box, covered with a chem wipe).
    1. There are variances in both time and temperature (some incubate rather than using room temperature), so this is something that could be varied if desired.
  3.  Aspirate formaldehyde and wash twice with PBS. Aspirate PBS.
  4. For measurements, fill with HBSS at 37C as is typical with live cell measurements.

Cell culturing protocol

Thawing Frozen Cells

  1. Cells arrive deep-frozen.
  2. Add cells to 10 mL of growth media with 20% FBS.
  3. Incubate overnight or until confluent.
  4. Passage to 2 dishes with 10 mL of growth media with 10% FBS
  5. Continue to passage about twice a week.

Passaging

  1. Aspirate (remove) media.
  2. Add 1 mL of trypsin, coating the entire bottom of the dish.
  3. Swirl for 20 seconds, then aspirate.
  4. Add 1 mL trypsin.
  5. Incubate for 4 minutes.
  6. Use the pipette to “pressure wash” cells off of one dish with trypsin.
  7. Add the trypsin with the lifted cells to the second dish.
  8. Use pipette to wash cells off of the second dish with trypsin.
  9. Add (x) μL of trypsin with lifted cells to new dishes with 10 mL of growth media.
    1. (x) should be somewhere between 75 and 150, depending on how quickly you want the cells to reach confluence

Cell Media/Solutions

Cell Media (EMT6)

  • 500 mL BME (Basal Medium Eagle. We have always gotten this from the Foster lab)
  • 2.5 mL Pen Strep
  • 2 mL Fungizone
  • 1 mL plasmocin
  • 1 mL primocin
  • 10% FBS – 55.5 mL (Fetal Bovine Serum)

*Note: 20% FBS media is also needed for when cells are first thawed.

*For SCC7 cell line, components are all the same except instead of BME base media, use RPMI media

*All Nada solutions are based on 500mL total final solution

Nada Solution #1 (basic cell measurement solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008  g D-glucose
  • 6 mL Hepes
  • 0.0832 g CaCl2
  • 0.0571 g MgCl2

Nada Solution #2 (permeabilizing solution)

  • 3.9447 g NaCl
  • 0.2199 g KCl
  • 0.9008 g D-glucose
  • 6 mL Hepes
  • 0.476 g MgCl2
  • 20 μM Ionomycin

Nada Solution #3 (calcium shock solution)

  • 3.9447g NaCl
  • 0.2199g KCl
  • 0.9008g D-glucose
  • 6mL Hepes
  • 0.0888g CaCl2