Lab3 Flashcards
(25 cards)
What solution is the piece of fresh pig spleen submerged in?
6 ml of ice-cold Alsever’s solution
What is the purpose of the cell strainer in the procedure?
To strain the contents of the pig spleen and release the cells into the Alsever’s solution
What tool is used to cut the spleen into small pieces?
Scissors
What is the term for the white cells isolated from the spleen?
Splenocytes
How do you indicate a significant number of splenocytes have been released?
The Alsever’s solution turns thick
What is the next step after labeling the 15 ml tube with Ficoll-Pague PLUS?
Layer 4 ml of the splenocyte suspension on top of the Ficoll-Pague reagent
What should you avoid doing when adding the splenocyte suspension to the Ficoll-Pague?
Do not shake or mix the two layers of media
At what speed and duration should the centrifuge be set for the first spin?
400 x g (1500 rpm) for 30 minutes at 18°C
What should be done after centrifugation regarding the top layer of the medium?
Remove and discard the top layer carefully
What is critical when isolating the splenocyte layer?
Transfer with a minimum amount of Ficoll-Pague PLUS and supernatant
What is the centrifuge speed and duration for the second spin?
100 x g (800 rpm) for 10 minutes at 18°C
What medium should the cell pellet be resuspended in?
6 ml RPMI culture medium
What should be labeled on the 6-well cell culture plate?
Your name and group number, and wells from #1 to #6
Fill in the blank: The splenocyte suspension is transferred into a 15 ml tube with _______.
10 ml PBS
True or False: The splenocyte layer should be disturbed when discarding the top layer of medium.
False
How should the wells be divided for the experiment?
The wells should be divided into 2 rows, each with 3 wells.
What is added to well #1?
In well #1, add 60 ul splenocyte suspension and 2.9 ml RPMI culture medium.
What is added to well #4?
In well #4, add 300 ul splenocyte suspension and 2.7 ml RPMI culture medium.
What is the composition of well #2?
In well #2, add 60 ul splenocyte suspension, 2.4 ml RPMI culture medium, and 0.5 ml LPS.
What is the composition of well #5?
In well #5, add 300 ul splenocyte suspension, 2.2 ml RPMI culture medium, and 0.5 ml LPS.
What is the composition of well #3?
In well #3, add 60 ul splenocyte suspension, 2.1 ml RPMI culture medium, and 0.8 ml LPS.
What is the composition of well #6?
In well #6, add 300 ul splenocyte suspension, 1.9 ml RPMI culture medium, and 0.8 ml LPS.
What is the incubation condition for the cells?
Incubate the cells at 37°C overnight in a CO2 incubator.
What should be done the next day after incubation?
Observe the cells under an inverted phase contrast microscope and take photos.
