Version+of+Chromium+Release+Assay+Protocol+for+Age+Associated+Stem+Cell+Autoimmunity+Experiment+11-23-12

Approximate Timing of Experiment (times are probably underestimated) 9-10am: perform splenectomy, obtain bone marrow, and perform cardiac puncture on mice 10-11:30am: isolate stem cells 11:30am-12:30pm: incubate target stem cells with radioactive chromium, and process spleens to obtain splenocytes 12:30-1:30pm: combine splenocytes and target stem cells in desired combinations and ratios 1:30-7:30pm: killing assay incubation 7:30-8:15pm: add supernatent to Lumaplate 8:15-overnight: air dry Lumaplate overnight next day: read Lumaplate in Topcount (takes about 1 hr)

//Materials:// >> 1 mM sodium pyruvate >> 1× nonessential amino acids
 * radioactive sodium chromate
 * target cells (stem cells from mouse)
 * complete RPMI-10 medium (antibiotics not necessary in medium used for this assay)
 * sensitization medium
 * Complete (so FBS) RPMI-10 medium (APPENDIX 2) containing:
 * Fetal bovine serum (FBS), heat-inactivated 1 hr at 56C
 * Effector cells (Including CTL)
 * 2% (v/v) Triton X-100 in H20
 * 100 uL used for each well of a few wells for the maximum/complete lysis control
 * last seen in radioactive tissue culture hood
 * mesh filters
 * 15mL conical tubes with screw caps
 * Multichannel pipettors (50 to 200 uL) with disposable tips
 * Lumaplates for solid scintillation counting (these plates can hold 100 uL; liquid should be added, and then the plate should be dried)
 * Additional material for preparing single-cell suspension and cell viability test using trypan blue exclusion
 * CountOff or Lift-Away (or just soap and water if you don't have these in a tight situation) for cleaning spilled radioactive material
 * TopSeal-A
 * next to Lumaplates on the top shelf of tissue culture room

//Part I - Prepare Target and effector cells//


 * Note: desired effector cell concentration 1*10^7 cells/mL. Also, the TopCount could be calibrated before (maybe a day before the experiment)
 * May want to start thawing FBS needed later.
 * Collect target stem cells. See Mouse Bone Marrow Isolation Protocol. The final dilution of the target cells can be in sensitization media.
 * Collect splenocytes. See Surgical Removal of Spleens
 * Incubate the target cells with chromium. Add 20 uL FBS per 100 uL (I'm not sure how necessary this FBS step is) and 0.2 mL (actually I will use about 0.1 mL for this experiment) of -1 mCi/mL 51Cr (ordered chromium often comes in containers containing 1 mCi total which water must be mixed with after the first opening of the bottle). Mix gently and incubate in a loosely capped 15-mL conical tube or 14 mL round bottom tube for about 1-2 hr at 37 C, 5% CO2 incubator. Start preparing the effector cells in the next steps during this incubation.
 * 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.
 * About how much radiation would the researcher be exposed to?
 * 1 mCi/mL*0.2*mL = 0.2 mCi
 * Gamma Ray Dose Constant for Chromium: 0.26 mR/hr per 1 mCi at 30 cm
 * Therefore exposure is about: 0.2 mCi*0.26 mR/(hr*1 mCi) at 30 cm = 0.052 mR/hr at 30 cm
 * The annual ALARA limit for the whole body is 5000 mR. Therefore, generally the total amount of radiation the researcher would be exposed to during this experiment would be fairly small.
 * Other notes about radiation
 * Although the best protection against gamma radiation is plastic followed by lead, even having more and thicker layers of plastic can greatly reduce the amount of gamma radiation that comes through
 * Note that currently the acrylic shielding in our lab does not contain lead. This is something that I can consider purchasing in the future. Alternatively, I could just use the lead sheets or lead/plastic sheets that we have now.
 * Raymon Maderas mentioned that I can block radiation from waste containers by placing lead shielding around the waste area.
 * Raymon thinks it is not necessary to try to place lead around the centrifuge when in use.
 * Raymon said that if I really wanted to that I could put a container containing radioactive liquid waste into the liquid waste container, but he would prefer that we just put liquid into the liquid waste.
 * Our stock sodium chromate container can be kept at room temperature in the "hot" room. It might be good to have some shielding around it. The door to the hot room should also be kept closed.
 * Used Lumaplates can be stored in one of the shielding boxes. May want to put lead shielding around the area as well.
 * The geiger counter pancake counts both beta and gamma radiation
 * The half-life of 51Cr is 28 days. Smaller volumes (e.g., 0.05 to 0.1 ml) of 51Cr solution maybe adequate for labeling cells. Tumor cells and cultured cell lines may label adequately with a specific activity of <200 µCi/µg. The volumes of all materials can be expanded if larger numbers of 51Cr-labeled target cells are required.
 * Gently resuspending cells once during incubation may enhance labeling.


 * Prepare effector cells (see Splenocyte Preparation Protocol)) (make sure red blod cell lysis buffer is used in your procedure), and then resuspend in complete RPMI-10 at 10^7 cells/mL


 * Dispense 0.1mL of effector cells to the wells of 96-well microtiter plates (flat bottom plate may be best), with replicates of 3 or 4 wells for each effector cell concentration.
 * “Effector cells” commonly refers to all cells in a suspension consisting of CTL and nonlytic cells. E:T refers to the ratio of the total number of effector cells (not just CTL) to target cells. A ratio of 10^6 effector cells to 10^4 target cells per well is represented by E:T = 100. Use the suspected level of CTL activity to calculate the range of effector cell concentrations. A small dose-response assay in which three or four different E:T ratios are used is a good starting point; this assay commonly includes replicates in which the E:T ratios are 100, 30, 10, and 3. Unusually active CTL can result in detectable lysis at effector:target ratios <1:1. Concentrations >2×106 cells/well commonly suppress activity.
 * 100 (100/1=10^6/y -> y = 10^6/100 = 10^4) (10^6 effectors to 10^4 targets)
 * 30 (3*10^5 effectors to 1*10^4 targets)
 * 10 (1*10^5 effectors to 1*10^ targets)
 * 3 (1*10^4 effectors to 1*10^4 targets)
 * may want to organize plate so that released chromium concentration would increase from the back to the front. This would be most pertinent if you plan to reuse the same tips from row to row which some protocols claim is acceptable.
 * possible controls
 * Target cells with 2% v/v Triton-X 100. May want to add triton before the long incubation, but some people do it afterward.
 * "For the control chromium release wells, add 0.1 mL of 2% Triton X-100 (lysing agent) to measure maximum releasable 51Cr to a replicate set of empty wells with 0.1 mL target cells and mix by pipetting."
 * Note that it may be best to put these triton controls in a totally separate plate since wells with very high reactivity in a Luma plate can affect the readings of nearby wells.
 * Target cells alone
 * effector cells alone
 * see Plate Map for Age Associated Stem Cell Autoimmunity Experiment 11-23-12
 * see Plate Map for Age Associated Stem Cell Autoimmunity Experiment 1-10-13


 * 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.
 * 51Cr labeled target cells should be added to effector cells as soon as possible after the last wash
 * 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.
 * Large tumor or tissue culture cells may incorporate sufficient 51Cr to be used at only 10^3 cells/well in the assay. By contrast, freshly explanted lymphocytes may incorporate the label poorly, so that >5×10^4 cells/well are needed. Generally, sufficient numbers of target cells should be added to each well to provide =1000 cpm above background counts from the liquid scintillation counter.

//Part III - Kill Target Cells with CTL//


 * 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:
 * 0.1 mL effector cells
 * 0.1mL control lymphocytes
 * 0.1mL medium (to measure spontaneous Cr-51 release)


 * Centrifuge plates for 30 seconds at 200Xg in a Sorvall H-1000B rotor with microtiter plate carrier to promote contact between effector and target cells. Incubate plates for 6 hours (3-6 hours can be used) in a humidified 37C, 5% CO2 incubator.
 * Effector cells will bind to target cells at room temperature. Target cell lysis will not begin until the temperature is increased to 37C.
 * The incubation time can vary markedly depending on the purpose of the experiment, the activity of the effector cells, and the susceptibility of the target cells to spontaneous 51Cr leakage. Under some conditions—e.g., with particularly robust target cells—overnight incubations are possible.


 * 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
 * TopSeal-P and TopSeal-S can be heated onto the plate with the Micromate Heat Sealer (see Packard TopCount Microplate Scintillation and Luminescence Counter Getting Started Manual pg. 45-47)

//Part IV - Determining CTL Activity//


 * 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. The lower calibration protocol (protocol number 10) 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.

> corrected % lysis = 100 * (test51Cr_released-control51Cr_released)/(maximum51Cr-control51Cr), > where test refers to effector cells with CTL activity and control refers to nonlytic cells (“control effector cells” in materials list) or cell-free medium.
 * Calculate corrected percent lysis for each concentration of effector cells, using the mean cpm for each replicate of wells:
 * Release of 51Cr from target cells incubated in medium alone is often referred to as “spontaneous release.” Alternatively, substitute the background count of the scintillation counter for “control 51Cr released” in the denominator. For a description of other controls necessary for determining CTL specificity, see Critical Parameters.
 * What are some good numbers for this protocol. This website may have some good information (especially in the references): http://perkinelmerreagents.onconfluence.com/display/ts/Chromium-51+release+assay
 * Present CTL data in lytic units, graphs, or lysis values (see Anticipated Results).
 * When reporting results, the range or upper limit of spontaneous 51Cr-release values should be mentioned along with standard errors of the % lysis values.
 * Follow radiation cleanup protocols.
 * Fill out radioactive disposal logs and tape them to waste disposal containers. Note that a new log does not need to be filled out after each experiment. The same log can be added to until the radiation safety office picks up the waste. Contact the radiation safety office to come pick up the waste.
 * logs on wall in radiation room.
 * An example radioactive waste disposal log here: "S:\Research\CTL\Radiation\example radioactive waste disposal log.jpg"
 * If they are notified by Wednesday they pick up on Thursdays and Fridays. If notified after that they bring by empty containers and pick up the waste on the following Thursday or Friday.
 * Fill out source utilization report form. This will require using the geiger counter to measure radiation in some of the areas where it was used.
 * An empty chromium stock container can be placed next to but not in the radioactive liquid and solid waste containers.
 * Measure radiation in the main locations where it might be used, and record the radiation levels observed in a spreadsheet for our lab.
 * "S:\Research\CTL\Radiation\Radiation Records.xlsx"
 * Note that dosimeters are measured quarterly. (see information about accessing dosimeter records)