Protocols Involving Dendritic Cells:
Infecting Dendritic cells with LP
In this experiment, we will infect dendritic cells which have been obtained from the bone marrow of mice. See on how to obtain such dendritic cells.
Prepare 5% RPMI without Antibiotic
Collect Your DC cells: (They are in the medium)
reagents: –prepare 10% RPMI without Antibiotic –pour some PBS into 50 ml tube and let warm up *in incubator for 10 minutes if want; needs to be room temp)
1) At this point your DCs have been in culture for 6-10 days.
2) Your cells will be a mixture of macrophages which adhere to the plate and dendritic cells (most of which do not adhere). Using pipette, siphone off the liquid being careful not to scrape and stire up the macrophages which are adhered to the place. Place liquid into 50 ml tube.
(3) Add about 1 ml (room temp) PBS to each well plate in order to wash the rest of DC. Room temp PBS will not lift the macrophages. Place liquid into your 50 ml tubes.
(4) spin at 10 C at 1100 RPM for 10 min.
(5) Remove supernatent
(6) resuspend cells and transfer to tube using 10 ml medium (use 10% RPMI but without antibiotic since antibiotic will inhibit the infectivity of Lp)
(7) count the cells as below. (always keep cells on ice while doing anything!)
Prepare LP:
reagents: –blank cuvette, sample cuvette, transfer pipette, 2×2 square of parafilm –also turn on machine so warms up
(5) Put saline (about 5 ml) into 15 ml tube. take a [this is a tube which has been taken from -80, streaks out onto agar and incubated for 48 hours) which is on special agar (BCYE). Use swab end to collect and put into your tube. Repeat with cotton swab if necessary. You should just barely see letter in back of tube. (if you do not see you can add more saline)
(6) take 1 ml of LP-solution and transfer to disposable cuvette. (place parafilm around cuvette!)
(7) take OD reading. get blank in drawer. put blank in. Change wave lenght to 620. change parameter so can print out. Convert reading using graph into number CFU. (write this on the tube!)
<Turn on> (takes 5 min) 1st place blank in. <1><enter>”change parameters” change the second alpha to “620” (the first stays the same at “800) <Y> autozero <start> (need to be at 0). Place sample <start> again (your reading should be close to 1 (e.g., .834 converts to 21×108 so write on tube 2.1 x 109) hit return and turn machine off
(8) ) take your DC cells out of centrifuge from (4) above and pour off supernatent into sink. Transfer pellets into 15 ml tube having about 10 ml RPMI (if not expecting a lot of cells can use 5 ml). (so you can pipette a 3 ml RPMI volume up and down in one 50 ml tube and do the same for the others to get that 10 ml volume. You will need to know this final volume for calculations below)
Count your DC cells:
(9) take 50 ul from your tube and add to 50 ul tryptan blue (you can do this in a well plate) Look under microscope low magnification 1st for grid. count 5 squares. (corners and middle)
# cell counted X 2 X 5 X 104 (for the cuvette) x 10 = your total # cells per ml
[Let say your total number is 20 x 106 per ml. We want a final concentration of 1 X 106 per ml. What volume should we resuspend our cells in to get this final concentration?
20 x 106 / X = 1 X 106 / 1 ml
x= 20 ml. So if our starting concentration was 10 ml we would need to add another 10 ml to get this final concentration. (use larger 50 ml tube to make this appropriate volume)
(10) Now your are ready to Transfect your cells.
Transfecting Cells:
(1) The infection ratio is 10:1. So figure out how much of your Lp you should use to infect your cells. Suppose you have 4 million cells. You would use 14 ul of LP. So spin your cells down (want to infect in about 1 ml), add 14 ul of LP and incubate at 37 C for 30 min.
Ex. you have 4 million cells and 2.8 x 109 cell/ml bacteria. How much LP should you add?
2.8 X 109 cells/ml (X) = 4.0 X 107 X=4.0 X 107 / 2.8 X 109 =1.4 X 10-2 = .014 ml = 14 ul of LP
Ex. From your OD reading you determine you have 2.3 x 10e9 cells/ml Lp. You have counted 5 million cells/ml of DCs. You u want an infection fatio DC:Lp of 1:10 how much Lp should you add?
2.3 X 109 cells/ml (X) = 5.0 X 107 X=21.7 ul
Remove Excess LP (wash the cells)
(1) add Hanks solution (HBSS) to the tubes (fill up the tubes with hanks). Always pour Hanks from the large stock flask into a smaller flask and from there into your tubes. Centrifuge for 10 min
(2) remove supernatent (make sure this goes into bleach since it is infected and let it sit for an hour!)
(3) repeat by filing tube with hanks, spin, and then removing supernatent into bleach
Resuspend pellet in appropriate amounts medium
(1) If, for example, you had 4 million cells in the pellet, you might add 4 ml media to get 1 million cells/ml. This will all depend on your experiment.