Ringers Solution, D. innubila Dissections, and Primary Cell Generation

20230215 Ringers Solution

  • Want to dissect the flies in ringers solution, it is not at the Biostore so I should make it myself
  • I used this recipe cut in half to make 500mL
  • I used this resource on how to make 1N HCl solution
  • I mixed these chemicals in 450mL of diH20:
    • 0.165g of Calcium Chloride dihydrate
    • 6.8g of Potassium Chloride
    • 1.35g of Sodium Chloride
    • 0.605g of Tris Base
  • Then I checked the pH with pH test strips. The pH is supposed to be ~7.2 and I am supposed to use HCl to titrate it down. This was kind of hard because I got a second brand of test strips (seemed more sensitive) and it have me different info than the other brand.
  • Because the other brand said the pH was perfect, I was inclined not to believe it. Plus I check the whatman indicator strips on di-water and it said that pH was 7, which I assumed was right. So I decided to trust the Whatman strips that said the pH was at 10
    • water pH:
  • I made 1N HCl by using our stock HCl that is 35-38%
    • 8.3mL stock HCl
    • 91.7mL Di-water
  • I did all of this in the hood wearing a lab coat, and made sure to add the acid to the water and mixed
  • Then I used a transfer pipette to drop in HCl to the ringers solution until the pH lowered to about 7, unfortunately I can’t completely tell how specific the pH is, and I might have gotten it a little under
  • Added the remaining 50mL of DI-H20
  • Mixed and vacuum filtered the solution through at 0.45um filter into a glass bottle
  • Autoclaved the bottle on setting 3 and placed it in the cell culture fridge to store afterwards

20230216 Ovary Dissection and Primary Cell Culture

  • Isolated out 50 females into empty vials, 5 flies per vial about an hour before starting so the would hopefully poop out much in their guts
    • Females were age “4”
  • Prepared:
    • a small beaker with room temp ringers solution
    • a dish with 70% ethanol
    • a dish with DI-water
  • Rob showed me how to do dissections, but all flies except 2 were done by me
  • Used microscope slides and dissecting forceps
  • Steps of each dissection:
    • Put a drop of ringers on the microscope stage
    • Placed a blank glass slide on the water drop
    • Added a drop of ringers to one side of the slide
    • Put one vial of flies to sleep on the CO2 pad
    • Took a fly with forceps and dipped it into 70% ethanol, and then into the DI water
    • Placed the fly on the droplet of ringers on the slide
    • Use the forceps to try to pull off some tergets on the abdomen of the fly while holding the thorax with the other forcep
    • Tried as best as possible to squeeze out the ovaries and not break the gut
      • This was tough, many flies just fell apart as I dissected, or had small ovaries that I had to dig through the gut to get to
    • Picked up the isolated ovaries and put time into the ringers solution beaker
    • Dipped the forceps in 70% ethanol
  • This was repeated for ~45 flies
  • Then the beaker with the ovaries was taken to the cell culture hood in 4012
  • There I had fly extract, mushroom extract, and mediums warmed to room temperature
  • Put a mesh 100um cell strainer in an autoclaved flask and poured the ovaries in through the mesh. To get any stuck on the beaker I used embryo wash to wash them into the strainer
  • Squirted 70% ethanol over the strained ovaries for about 1 minute to try to sterilize them
  • Washed the ovaries out into a 50mL conical with 7mL of 10% FBS Schneider’s medium
  • Put that liquid into a 10mL glass tube and let the ovaries settle (did not need to centrifuge)
  • Removed all the liquid from the 10mL tube
  • Washed the ovaries again with 7mL of 0% FBS Schneider’s medium
  • Removed all the liquid from the 10mL tube
  • Added 3mL of 10% FBS Schneider’s medium
  • Transferred the liquid and ovaries to the 7mL dounce homogenizer
  • Homogenized the ovaries as thoroughly as possible
  • Moved the homogenate to a new T25 flask with 16mL of 10% Schneider’s medium
  • Put 9mL of that liquid into a new T25 flask
  • Added 1mL of fly extract to the flask with 9mL ovary homogenate
  • Added 400ul of mushroom extract to the flask with 10mL ovary homogenate
  • Placed both flasks in the 23C incubator