LABS

Question 1

what does number of cells (cells) divided by the volume (mL) give you?

Answer 1

concentration (cell/mL)

Question 2

what does concentration (cells/mL) multiplied by volume (mL) give you?

Answer 2

number of cells (cells)

Question 3

what does number of cells (cells) divided by concentration (cells/mL) give you?

Answer 3

volume (mL)

Question 4

what does the concentration you want (want) divided by the concentration you have (have) in your stock suspension multiplied by volume of cells required

want/have x volume of cells = ?

Answer 4

the amount to add to get your desired working solution (conc. you want for experiment)

Question 5

want and have values are ________ and must have the same _______ for working and stock solutions?

Answer 5

concentrations

units

Question 6

we have 12mL stock cell suspension at 10 cells/mL and would like 5mL of working solution at 2 cells/mL

how much of your stock solution would you add to get to the working solution?

Answer 6

2/10 x 5

= 1mL

so you add 1mL of stock suspension to 4mL of diluent to get a 5mL working solution at 2 cells/mL

Question 7

in the equation C1V1 = C2V2 which one is the stock solution and which is the working solution?

Answer 7

C1V1 = stock solution conc. and volume

C2V2 = working solution conc. and volume

Question 8

we have a 12mL stock cell suspension at 10 cells/mL and we want to make 5mL of working solution at 2 cells/mL

how much of your stock solution would you add to get to the working solution?

Answer 8

2x5/10

= 1mL

added to 4mL diluent to make 5mL working solution at 2 cells/mL

Question 9

what is trypan blue used for?

Answer 9

selectively staining dead cells blue allowing us to identify the live ones with membranes that expel the stain

Question 10

how do you calculate the number of cells per mL from a cell count in a haemocytometer?

Answer 10

N x dilution factor x 10^4

if you counted 47 and 62 and the dilution factor is 10:

N = (47+62)/2
dilution factor = 10

= 5.45 x 10^6 cells/mL

Question 11

you have a stock solution of cytokine IL-12 at a concentration of 40ug/mL. You need to prepare 5mL of a working solution of IL-12 at 2ug/mL

what volume of IL-12 stock solution do you need to take?

hint: want/have = amount to add

Answer 11

want/have = amount to add

2/40 x 5 = 0.25mL

so you need to take 0.25mL IL-12 stock solution and add to 4.75mL of diluent to get a total of 5mL working solution of IL-12 at 2ug/mL

Question 12

we have 12mL stock suspension of cells at 10 cells/mL and we want to make a working solution suspension of 5mL at 2 cells/mL

what volume is is required?

Answer 12

C1V1 = C2V2

10 x V1 = 2 x 5

V1 = 2 x 5 / 10 = 1mL

add 1mL to 4mL diluent

the 12mL was a distraction and we don’t actually need it in the calculation

or use logic:

2 cells/mL is one fifth of 10 cells/mL therefor you’ll need to dilute the stock suspension five-fold and one fifth of 5mL is 1mL

Question 13

why do we add anti-CD3 to our cell culture system?

Answer 13

activates the TCR (bypass signal 1)

Question 14

why do we add anti-CD28 to our cell culture system?

Answer 14

provides co-stimulatory signal (bypass signal 2)

Question 15

why do we add IL-2 to our cell culture system?

Answer 15

promotes proliferation

Question 16

how do we examine for cytokine and transcription factor production in our T cells?

Answer 16

using an intracellular staining assay and flow cytometry analysis

Question 17

how do we measure for cytokine production in our T cells?

Answer 17

using an ELISA assay

Question 18

what is the polarising cytokine for Th1?

Answer 18

IL-12

Question 19

what is the polarising cytokine for Th2?

Answer 19

IL-4

Question 20

what is the polarising cytokine for Th17?

Answer 20

TGFbeta and IL-6

Question 21

why do we add PMA, ionomycin and brefeldin when setting up T cell stimulation for intracellular cytokine staining?

Answer 21

PMA and ionomycin provide a form of non-specific T cell activation

brefeldin is a golgi transport inhibitor and will prevent cytokines from being secreted outside the cells

Question 22

what do fixation buffers (like those we added to cytokine staining and TF tubes) do?

Answer 22

stabilises proteins so that they are retained in their original cellular location and don’t ‘leak out’ once the cell is permeabilised

transcription factor fixation buffer acts to permeabilise the nuclear membrane so that antibodies can reach transcription factors in the nucleus

Question 23

what does perm buffer do?

Answer 23

permeabilises the cell membrane to enable fluorescent antibodies to enter the cells and bind to cytokines or transcription factors

Question 24

you have a tube containing 3mL of cells at conc. of 6x10^6 cells/mL and want to dilute to conc. of 4x10^5 cells/mL. What volume RPMI growth media do you add to tube?

Answer 24

C1V1=C2V2

6x10^6/4x10^5 x3

= 45mL

so you add 42mL to 3mL to get a conc. of 4x10^5 cells/mL

Question 25

you have prepared a tube of cells and take a sample for counting, making a 2-fold dilution in trypan blue. When you count this on the haemocytometer you get 63 and 71, what is the concentration of your cells?

Answer 25

(63+71)/2 = 67

C = 67 x 2 x 10^4

= 1.34x10^6 cells/mL

Question 26

a 12mL tube of cells contains 7.5x10^5 cells. You require a cell concentration of 5x10^5 cells/mL for an assay. What volume of cells can you prepare?

Answer 26

C = T/V

7.5x10^5/12 = 6.25x10^4

C1V1=C2V2

V2 = 6.25x10^4/5x10^5 x 12 = 1.5mL

Question 27

someone has a tube containing 14mL of cells and they dilute a 10ul sample of cells with 90ul of trypan blue and count them, coming out with 46 and 41. What is the total number of cells in the tube? What is the cell concentration?

Answer 27

46+41/2 = 43.5

C = 43.5 x 10 x 10^4

= 4.35x10^6 cells/mL

T = CV = 4.35x10^6 x 14 = 6.09x10^7 cells

Question 28

you have a stock solution of cytokine IL-12 with a conc. of 50ug/mL. You need to prepare 5mL of a working solution of IL-12 at 2ug/mL, what volume of IL-12 stock solution do you need to take?

Answer 28

2/50 x 5

= 0.2mL

Question 29

for an assay you want to plate out cells into 8 wells of a micro-titre plate, with each well containing 1.5mL of cells at a conc. of 1x10^6 cells/mL. What is the total number of cells you will need?

Answer 29

8x1.5 = 12mL

T = CV = 1x10^6 x 12

= 1.2x10^7 cells

Question 30

a tube contains 5mL of cells. You take a 20ul sample and make a 50:50 dilution of cells in trypan blue and count 81 and 67. How many mLs of cells at a conc. of 1x10^5 cells/mL could you make?

Answer 30

(81+67)/2 = 74

C = 74x2x10^4 = 1.48x10^6

C1V1=C2V2

V2 = 1.48x10^6/1x10^5 x 5

= 74mL

Question 31

you are given 100ul of anti-CD3 antibody stock solution which is at a conc. of 1mg/mL. You need to make up 4mL of media containing 20ug/mL anti-CD3. How much anti-CD3 stock solution will you need to take?

Answer 31

20/1000 = 0.2mg/mL

0.2/1x 4 = 0.08mL = 80ul