Detach and Freeze FB cells

What is the detaching reagent for the ImHUVEC cells?

0.05% trypsin with EDTA

What does the trypsin do?

Digest the protein used for the cells to adhere to each other and the surface of the flask

What happens if the trypsin enzyme is active for too long in the flask?

It can damage the cells

what is the inhibitor of the trypsin?

10% FBS

What media is used to culture the FB cells?

FibroLife

What is the neutralizing buffer labeled with?

An "N"

what is the neutralizing buffer? X

10% FBS with DMEM

What does the neutralizing buffer do?

stops the trypsin reaction

what do you do after prepping the BSC?

aspirate the media from the flask

what comes after aspirating the original media?

use PBS to wash the cells and any FBS residue

What can residual FBS do?

Inhibit the trypsin

what do you do after washing the cells with PBS?

aspirate the PBS

What do you do after aspirating the PBS?

Add the trypsin to the flask

what comes after adding trypsin to the flask?

put the flask in the incubator for 30 seconds

Why do you put the flask in the incubator?

To let the trypsin work at its optimal temp

What temp is the incubator set at?

37°C

why is the flask put in the incubator for only 30 seconds? XX

EC cells are relatively easy to detach

Do cancer cells and fibroblasts need more time to detach than EC cells?

yes

What happens if EC cells are treated with trypsin for too long?

they die

what do you do after taking the flask out of the incubator?

add the neutralizing buffer

what happens after you add the neutralizing buffer to the flask?

collect the cells in the buffer media to transfer them to a 15mL centrifuge tube

why do you centrifuge the cells?

to collect them as a pallet

What happens after transferring the cells in the neutralizing buffer to a 15mL tube?

centrifuge them for 5 mins at 1.2 rpm

after centrifuging the cells, what comes next?

aspirate the supernatant from the tube

What do you do after aspirating the supernatant?

resuspend the cells with bambanker and mix them

what do you do after resuspending the cells?

transfer them into the cryotube

What do you do after transferring the cells into the cryotube?

put the cryotube in Mr. Frosty

Where do you put the Mr. Frosty at the end?

in the -80 freezer

how long does the tube stay in the -80 freezer?

overnight

what do you do after placing the cryotube in the -80 freezer?

put it in the LN tank the next day

What passage number are the cells?

P8

What kind of cells are they?

Shun FB

what size dish are they cultured in right now?

10cm

how much of P8 will be frozen?

2/3

how much of P8 will continue to be cultured?

1/3

from P8 to P9 how are the cells split?

into thirds

what will happen to the 2/3 that will be frozen?

they will become P9

what will happen to the 1/3 that will continue to be cultured?

it will become P10

why do you only need 1/3 to culture?

1/3 of the cell passage can proliferate into 1 whole portion again

when do you know to split up the cell passage? (freeze or continue culturing)

when the cells become confluent

how much media of FB cells will there be?

6mL

why will there be 6mL of FB?

2mL for the trypsin to detach the cells, 4mL for the neutralizing buffer

how do you split the cell passage up?

4mL for freezing into P9, 2mL for culturing into P10

why do you split up the cell media into 4mL and 2mL?

because 2mL is 1/3 of the cell passage

when do you split up the cells? X

after adding the neutralizing buffer

How much BamBanker do you use to freeze one portion of FB?

100uL

how much BamBanker do you need total for freezing the FB cells?

200uL

Why do you need to use 200uL of BamBanker to freeze the FB cells?

Because two portions are being frozen

how do you label the cryovials?

cell type, passage #, volume of dish used

what tip do you use when freezing the cells?

1mL micropipette tip

why do you use a 1mL tip and not a 200uL tip?

there will be more liquid than the amount of BamBanker used

what size dish do you continue to culture the P9 cells?

10cm

How much PBS do you use to wash the cells? X

4mL? 8mL?

How much trypsin do you use to detach the FB cells?

2mL

How much neutralizing buffer do you use with FB?

4mL