BIOE 202 Lab practical

The steps/protocol questions are for a 6-cm plate specifically. This can also act as an overall review for lab 5's content

6 cm plate plating volume

5 mL

10 cm plate plating volume

10 mL

15 cm plate plating volume

20 mL

6-well plate plating volume

2 mL/well

24-well plate plating volume

.5 mL/well

96-well plate plating volume

.1 mL/well

Amount of trypsin and PBS wash (each)

½ of recommended plating volume

What do you do after taking cells out of incubator, before putting them in the BSC?

Check the cells under microscope - record confluency.

steps after initial observation

Aspirate media, then add 2.5 mL PBS & aspirate (make sure to not touch pipet tip to cells)

What is the purpose of adding and aspirating PBS?

Washing the cells - getting rid of any excess media

Step after PBS

Add 2.5 mL trypsin, incubate for 5 minutes

Why do we add & incubate with trypsin?

Trypsin helps the cells lift from the bottom of the plate/well, and it needs incubation to work.

What happens if we incubate for too long with trypsin?

it will damage & kill the cells.

Step after trypsin incubation

Look at cells under microscope to ensure cells are lifted. if they are not, incubate again for 2-3 minutes

Step immediately after you confirm cells to be lifted?

neutralize trypsin with 2.5 mL DMEM.

2 important (mini) steps after neutralizing with DMEM

1. pipet up & down to ensure cells are not clumped

2. DO NOT ASPIRATE - cells are suspended in solution

Step after neutralizing with DMEM

Transfer cells from plate to conical tube, and centrifuge at 1000 rpm for 5 mins

step after centrifuging

aspirate supernatant - DO NOT get too close to pellet

step after aspirating supernatant

resuspend pellet:

1. add 1 mL DMEM with p1000 and pipet up & down to resuspend

2. add 3 more mL DMEM with serological pipet & mix again

step after resuspending cells

mix 20 µL cell suspension with 20 µL trypan blue

What is important to note about trypan blue?

everything that touches trypan blue must go to separate trash container

How do you prepare the hemocytometer/glass slide?

thoroughly spray with IPA (kimwipe underneath). make sure to remove as much trypan as possible

How much of the trypan/cell suspension mix do you pipet into the hemocytometer?

10 µL

which edges do you count when counting cells with the hemocytometer?

top and right edges

What does a dead cell look like under a hemocytometer?

blue (live cells are yellow/white)

Formula for calculating cell concentration

(cells counted)*(2 / 4) * (10,000)

Formula for calculating volume of cell suspension to replate

(cell concentration)*(volume) = (number of cells to plate)

step after calculating volume of cell suspension to replate

1. add that volume of cell suspension to new 6-cm plate

2. add DMEM up to recommended plating volume.

step after replating cells

look at them under the microscope to make sure they are present and look healthy

Components of DMEM

FBS (fetal bovine serum - nutrient supplement), DMEM (everything a cell needs to grow), P/S (Penicillin/Streptomycin - to prevent unwanted bacterial growth)

What type of mammalian cells are we using?

HCT116