Chromium+Release+Test+10-18-12

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.

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.
 * 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
 * I think I would like to have the following samples (see plate map below for abbreviation details


 * 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.
 * 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
 * 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.
 * 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.
 * 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.

Plate map: "C:\kurt\storage\CIM Research Folder\DR\2012\10-18-12\cr test\cr test plate map 10-18-12.xlsx"
 * 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:
 * 0.1mL control lymphocytes
 * 0.1mL medium (to measure spontaneous Cr-51 release) or Triton depending on sample


 * 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.
 * 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.
 * 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.
 * 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)
 * 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.