Chromium Release Test 10-18-12

2015-01-13

azim58 - Chromium Release Test 10-18-12

I would like to test how several variables affect the radioactive
readings of target cells. I would like to see how the incubation time,
incubation volume, and flask type affect the radioactive readings of the
target cells. I will try 2 conditions for each of these variables.

Incubation time: 1 hr, and 4 hr
Incubation Volume: 500 uL, and 5 mL
Flask type: round bottom tube, tissue culture flask, and tissue culture
flask without any chromium just for cell counting

Here's a basic outline of the protocol I plan to follow

prepare a single-cell suspension of target cells in complete RPMI-10
medium.

* Resuspend cells in 5mL complete RPMI-10.
* Determine viable cell count of unsettled filtered cells by Trypan
  blue exclusion.

* Determine the amount of cells I want to pulse for each sample. For a
  normal test I place 1e4 target cells in each well of a 96 well plate. I
  think it would be good to pulse enough target cells for at least 4
  plates. Therefore, I would need to pulse a minimum of 4*96*1e4 = 3.84e6
  o I think I would like to have the following samples (see plate map
    below for abbreviation details

1hrR0.5mL
1hrR5mL
4hrR0.5mL
1hrTCF5mL
1hrTCF5mL no chromium
If I did 3.84e6 cells for each sample, then I would need 3.84e6*5 =
1.92e7 cells total.

* Add 20 uL FBS per 100 uL (I'm not sure how necessary this FBS step
  is) and 0.1mL of -1 mCi/mL 51Cr. Mix gently and incubate in a loosely
  capped 15-mL conical tube or 14 mL round bottom tube or a tissue
  culture flask depending on the sample type. Incubate for 1 or 4 hr
  (depending on the sample) at 37 C, 5% CO2 incubator.
  o Note that after chromium has been used, the use should be
    recorded on the Source Utilization Report Form on the wall in the
    "hot" room
  o Note that when working with chromium the proper radioactive
    shielding should be used (e.g. plastic shielding followed by a lead
    brick to shield the area from your body (lead stops gamma rays, but
    when beta particles hit lead they can produce Bremsstrahlung X-rays
    so it is best to protect with plastic first and then lead)). It is
    also probably a good idea to double glove in case any radioactive
    material spills onto your glove.
  o Gently resuspending cells once during incubation may enhance
    labeling.

* After the incubation determine the cell count of the cells in the
  tissue culture flask without chromium to see if the cells adhered
  within 1 hr.

* After the incubation with 51Cr, wash 51Cr labeled target cells 2 to 3
  times with 10 mL complete RPMI-10 as in step 2 (tube should be capped
  in the centrifuge). This can be done by taking the supernatent away and
  collecting the supernatent in a radioactive waste container. Resuspend
  labeled target cells in complete RPMI-10 to 10^4-10^6 cells/mL
  (10^3-10^5 cells/(100 uL)) (Note that this is most likely not the
  original volume that the cells were suspended in!). The best
  concentration to choose will depend on how well the cells incorporate
  the label. Quickly proceed to the next step.
  o Note that a glass transfer pipette can be used to remove the
    radioactive supernatant, but this glass transfer pipette should be
    discarded in the solid radioactive waste container with the other
    solid radioactive waste instead of the normal glass tissue culture
    waste container.

* Add 0.1 mL of 51Cr labeled target cells to replicate wells containing
  one of the following, for a final volume of 0.2 mL/well:
  o 0.1mL control lymphocytes
  o 0.1mL medium (to measure spontaneous Cr-51 release) or Triton
    depending on sample

Plate map: "C:\kurt\storage\CIM Research Folder\DR\2012\10-18-12\cr
test\cr test plate map 10-18-12.xlsx"

* Centrifuge plates for 5 minutes at 200Xg. Harvest ~0.1 mL of each
  supernantent and add supernatents into each well of a Lumaplate. Make
  sure that tips are firmly attached to the multichannel pipette, and
  that a a full 100 uL of each sample is transferred to the plate.
  o With a high-quality multichannel pipettor and firmly attached
    tips, it is not necessary to change tips between replicate wells.
    Carryover of sample is negligible, particularly if pipetting proceeds
    from replicates of low-level, cell-free 51Cr to those with high
    levels.

* Air dry or oven dry the luma plate, do not exceed 60 C.
  o The plates can be dried in the 55 C incubator in the radiation
    room. Drying takes about 2 hours, and you may want to do dry
    overnight.

* Seal the plate with TopSeal-A

* Count 51Cr in TopCount instrument.
  o The TopCount could be calibrated before the experiment if
    desired. Load calibration plate kept on the cart of the TopCount for
    this. Access protocol program definitions with F1. The higher up
    normalization or calibration protocol in the software (around number
    4) seems to just count the bottom row. I think the lower calibration
    protocol counts the pattern of wells in the plate. Both protocols
    take a while for the machine to perform (maybe 30 min-1hr or even
    longer)
  o To count a chromium plate, press alt+F2. Choose the chromium
    protocol (you'll probably have to look up the number in the protocol
    definitions with F1 on the original screen). Load and count the plate
    with the appropriate button as specified on the screen (make sure
    that the Stacker option is off if there is no stacker). Counting
    takes about 1 hr.

===========================================================================
Results

Images of computer screen with counts of plate
C:\kurt\storage\CIM Research Folder\DR\2012\10-19-12\CRA

Analysis
"C:\kurt\storage\CIM Research Folder\DR\2012\10-19-12\CRA\cr test
analysis 10-19-12.xlsx"

I don't think any solid conclusions can be made from this experiment
since the 10 uL chromium wells had cpm numbers that were so high that
they even affected the cpm numbers of surrounding wells. Therefore, it is
difficult to tell how much of an affect the 10 uL chromium wells had on
the readings of other samples. At least one conclusion that can be made
is that the tissue culture flask is not good to incubate the cells in
since there was no difference between the triton sample and the non
triton sample with this type of flask. Smaller volumes may be better for
the chromium incubation than larger volumes. One might conclude that a 4
hr incubation is better than a 1 hr incubation, but the 4 hr sample was
also closest to the 10 uL chromium sample so it is difficult to make any
conclusion.

Note that a simple trypan blue count of the target cells also revealed
that they do adhere to the flask in one hour.

In the future, when incubating the target cells with chromium, I think it
would be best to gently shake the sample occasionally to enhance chromium
labeling.

azim58wiki: