Difference between revisions of "BV-2"

Latest revision as of 13:00, 7 July 2023

Cell Culture Conditions

DMEM 4.5 g/L glucose supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. FBS is heat-inactivated by incubation at 56°C during 30 minutes.

Preparation for the passaging

Before you get started, make sure to put on gloves and lab coat, and spray hands generously with ethanol.

To prepare before passaging:

1.       Take out trypsin and GM with heat inactivated FBS, and place it into a bead bath.

2.       Turn on the lab hood and spray with tissue paper drenched in ethanol.

3.       Take out a few 5- and 10-mL long pipette tips from the drawer and spray with ethanol before placing into the lab hood.

4.       Spray the Vacusafe with ethanol generously before placing it into the lab hood.

5.       Take out two 50 mL Falcon tubes – spray with ethanol and place into the lab hood. Mark one of the Falcon tubes with WT, and the other with KO.

6.       Take out two big T75 flasks for cell culturing.

Passaging protocol

Spray your hands generously with ethanol before proceeding onto the next steps. Work with one flask at a time. Start with WT culture flask.

1.       Take out the culture flask with WT cells from the incubator and observe them under the microscope. Make sure that there are no suspicious looking things inside of the flask, or any weird discolorations.

2.       Place the culture flask with WT cells into the lab hood. Make sure not to spray the flask with a lot of ethanol – you do not want the ethanol to get inside of the flask.

3.       Use a 10 mL pipette tip to suck up the old growth medium from the culture flask. Discard the old GM into one of the 50ml Falcon tubes marked with WT. Set the WT Falcon tube aside into a holding tray.

4.       Wash the culture flask with 8ml PBS. Aspirate the PBS with Vacusafe and a glass Pasteur pipette.

5.       Add 1.5 mL Trypsin into the culture flask.

6.       Incubate the culture flask for 5 minutes.

7.       Take out the culture flask from the incubator and add 8.5 ml of fresh GM with heat inactivated FBS into it. Now you have 10 mL in total of trypsin with cells + GM in your culture flask.

8.       Pipette out these 10 mL of trypsin with cells + GM from the culture flask and add it to the WT Falcon tube with the old GM. You should now have a total of 20 mL worth of cells with GM in the Falcon tube. Set the WT Falcon tube aside in the holding tray in the lab hood.

Now, do the same for the KO culture flask:

1.       Take out the culture flask with KO cells from the incubator and observe them under the microscope. Make sure that there are no suspicious looking things inside of the flask, or any weird discolorations.

2.       Place the culture flask with KO cells into the lab hood. Make sure not to spray the flask with a lot of ethanol – you do not want the ethanol to get inside of the flask.

3.       Use a 10 mL pipette tip to suck up the old growth medium from the culture flask. Discard the old GM into one of the 50mL Falcon tubes marked with KO. Set the KO Falcon tube aside into a holding tray.

4.       Wash the culture flask with 8mL PBS. Aspirate the PBS with Vacusafe and a glass Pasteur pipette.

5.       Add 1.5 mL Trypsin into the culture flask.

6.       Incubate the culture flask for 5 minutes.

7.       Take out the culture flask from the incubator and add 8.5 ml of fresh GM with heat inactivated PBS into it. Now you have 10 mL in total of trypsin with cells + GM.

8.       Pipette out these 10 mL of trypsin with cells + GM from the culture flask, and add it to the KO Falcon tube. You should now have a total of 20 mL worth of cells with GM.

Now, you should have two 50 mL Falcon tubes, one for the WT cells and one for the KO. They should each have roughly 20 mL of fluid in them.

1.       Turn on the centrifuge. Turn up the knob on the centrifuge – it should set the centrifuge to “5” on the left hand side and “3” on the left hand side. Open the centrifuge, and place the two 50 mL Falcon tubes with WT and KO cells into the big slots inside of the centrifuge. Close the centrifuge and let it do its job for 5 minutes.

2.       Once the centrifuging is over, take out the Falcon tubes and spray them with ethanol before placing back into the lab hood.

Now, work with one Falcone tube at a time. As you will be able to see, there will be a white cell pellet at the bottom of the Falcon tube. Those are your cells.

Start with WT:

1.       Aspirate the Falcone tube with WT cells using the Vacusafe and a pasteur glass pipette. ASPIRATE SLOWLY – MAKE SURE NOT TO SUCK UP THE CELL PELLETT AT THE BOTTOM OF THE FALCON TUBE!!!!!! LEAVE SOME MEDIUM AT THE BOTTOM OF THE TUBE – DO NOT TRY TO SUCK UP EVERYTHING!!!

2.       Once most of the medium is aspirated, add 10 mL of fresh heat inactivated GM into the Falcon tube with the WT cell pellet at the bottom. Mix well.

3.       Add 1 mL of the cell mix into a new T75 culturing flask.

4.       Top it off with 9 mL fresh GM.

5.       Mark the culturing flask with the name of the cells (BV-2 WT), the date, and your initials. Add the splitting ratio (1/10).

Now, move onto the KO:

6.       Aspirate the Falcon tube with the KO cells using the Vacusafe and a pasteur glass pipette. ASPIRATE SLOWLY – MAKE SURE NOT TO SUCK UP THE CELL PELLETT AT THE BOTTOM OF THE FALCON TUBE!!!!!! LEAVE SOME MEDIUM AT THE BOTTOM OF THE TUBE – DO NOT TRY TO SUCK UP EVERYTHING!!!

7.       Once most of the medium is aspirated, add 10 mL of fresh heat inactivated GM into the Falcon tube with the KO cell pellet at the bottom. Mix well.

8.       Add 2.5 mL of the cell mix into a new T75 culturing flask.

9.       Top it off with 7.5 mL fresh GM.

10.   Mark the culturing flask with the name of the cells (BV-2 KO), the date, and your initials. Add the splitting ratio (2.5/10).