Chromium+Release+Assay+Protocol

__**Chromium Release Assay for Measuring CTL Activity**__

As described in “Assays for T Cell Function” from Current Immunology Protocols, contributed by Wonderlich, Sheared, Livingstone and Brooks. "C:\kurt\storage\CIM Research Folder\DR\2013\4-13-13\download\Assays for T cell function from current protocols in immunology.pdf" Here's just the 51Cr part of the CTL assay document "F:\kurt\storage\CIM Research Folder\DR\2013\6-21-13\wiki_download\Chromium Release Assay.pdf"

B16 Current Protocols in Immunology also has protocols to test response of T cells to tumor cells pg. 20.1.13-20.1.16 (see "C:\kurt\storage\CIM Research Folder\DR\2013\4-13-13\download\B16 model protocol-1.pdf")

Example Approximate Timing of Experiment 9am work with target cells 10am start peptide pulse incubation 10-11am perform splenectomy and cardiac puncture on mice 11am prepare target cells for radiation uptake 11:15am-1:15pm incubate target cells with radioactive chromium, and process spleen 1:15pm combine effectors and targets 1:15-7:15pm killing assay incubation (may last less time or even go overnight depending on situation and potency of effector cells for targets) 7:15-8:45pm add supernatent to Lumaplate 8:45-10:45pm air dry Lumaplate (may want to do this overnight) 10:45pm read Lumaplate in Topcount (takes about 1 hr, may want to leave the plate overnight and obtain the results the next day)

//Materials:// >> 1 mM sodium pyruvate >> 1× nonessential amino acids > >
 * radioactive sodium chromate (see Ordering Chromium)
 * ~1mCi/mL Na2CrO4 in isotonic medium, sterile and pyrogen-free (200 to 500uCi/ug; DuPont NEN or Amersham)
 * target cells (e.g., single-cell suspension of splenic lymphoblasts, tissue culturecells, or tumor cells)
 * control target cells (matched to test target cells except for differences in antigen expression)
 * complete RPMI-10 medium (antibiotics not necessary in medium used for this assay)
 * sensitization medium
 * Complete (so FBS) RPMI-10 medium (APPENDIX 2) containing:
 * mitogen solution (optional for preparing splenic lymphoblasts): 1mg/mL Con A in PBS or 1mg/mL LPS in water (to simulate T and B cells, respectively),
 * Fetal bovine serum (FBS), heat-inactivated 1 hr at 56C
 * Effector cells (Including CTL) (Note that the T cells could come from a 5 day induction or straight from the spleen)
 * Control effector cells (nonsensitized spleen cells or spleen cells sensitized against irrelevant antigen)
 * 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
 * 25cm^2 tissue culture flask
 * 24-well flat-bottom microtiter plates, 2mL capacity with lids (this was in the original protocol, but I don't think it is necessary)
 * mesh filters (original protocol: nylon filtration fabric, 112-uM mesh (optional; recommended company: Tetco))
 * 15mL conical tubes with screw caps
 * Sorvall H-w000B rotor (or equiv) and microtiter plate carrier
 * multiwick supernanent harvesting system (Skatron) (this was in the original protocol, but I don't think it is necessary)
 * 96-well round-bottom microtiter plates with lids to fit supernanent harvesting system (Costar) (this was in the original protocol, but I don't think it is necessary)
 * 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)
 * Yvette used Scint 20 with Luma plates.
 * 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
 * antigen or peptide if needed for pulsing target cells (this would depend on the experiment)
 * last seen at tissue culture -20 freezer
 * Note that DMSO alone kills the target cells! Use as little DMSO as possible or even no DMSO.
 * see SIINFEKL peptide inventory

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


 * Note: desired effector cell concentration 1*10^7 cells/mL. Also, the TopCount could be calibrate before (maybe a day before the experiment)
 * May want to start thawing FBS needed later. May want to skip to collecting the effector cells if you would like to pulse the target and effector cells at the same time.
 * prepare a single-cell suspension of target cells in complete RPMI-10 medium.
 * The final dilution of the target cells can be in sensitization media.
 * If splenic lymphoblasts are to be used, preactivate (in vitro) ~5 × 106 spleen cells/ml in ~2 µg/ml Con A or -10 µg/ml LPS (both stocks diluted in sensitization medium) for stimulating T cells or B cells, respectively. Incubate spleen cells 2-3 days in a 25cm^2 upright tissue culture flask or 2mL flat bottom microtites plate in a humidified 37C, 5% CO2 incubator. <-Note that it may not be necessary to do this. It may be possible to use the target lymphoblast cells directly.
 * Here are some other notes from the original protocol
 * Lymphoblasts, tissue culture cells, or tumor cells are recommended as sources of target cells because they take up more 51Cr with less subsequent spontaneous leakage than nonactivated, freshly explanted normal cells. If lymphoblasts are used, they must first be activated as described above. If tissue culture cell lines or tumor cells are used, they must be of the appropriate MHC phenotype; often-used tumor cells are P815 (H-2d), EL4(H-2b), and LK (H-2k), all available from ATCC.
 * An alternative to collecting spleens for stimulator cells (see Basic Protocol 1, step 2) on day 0 and again 2 to 3 days later for target cells is as follows. Collect spleens for both stimulator and target cells on day 0. Place tightly capped culture flasks for target cells at 4 C; 2 to 3 days later add mitogen as noted in step 1, loosen cap, and transfer to a CO2incubator for 2 to 3 days.
 * Control target cells should not express the antigens used for sensitization and should be matched as closely as possible to the test cells, except for differences in antigen expression.Control cells are also prepared in complete RPMI-10.
 * Transfer cells to 15mL conical tube and wash cells once in 14mL complete RPMI-10 by centrifuging for 5 minutes in ~200Xg (1000 rpm in Sorvall H-1000B rotor) at room temp. Discard the supernatant.
 * Resuspend cells in 5mL complete RPMI-10. Allow cell aggregates to settle by gravity for several minutes or pass cell suspension through a single layer of 112uM mesh nylon (40 or 70 um or other lengths would probably also work) filtration fabric resting on the open top of a 15mL conical tube (press a pipet tip into the nylon fabric to form a small funnel). If a funnel is difficult to form with a 15-ml conical tube, a 50-ml tube can be used.
 * Determine viable cell count of unsettled filtered cells by Trypan blue exclusion.
 * Target cells should have a high viability (>80%). Ficoll-Hypaque gradient centrifugation (UNIT 3.1) can enrich the population of live cells, but live cells isolated from a starting population with poor viability generally leak 51Cr more than cells from a starting population with high viability.
 * (Note that I may want to consider revising this part of the protocol because it may be better to incubate with chromium first and then pulse some of the cells with peptide and don't pulse some of the other cells. This way the peptide pulsed and non-pulsed groups would have been labelled with the same amount of chromium) -> Pulse target cells. However, before pulsing the target cells use some cells for more tissue culture and use some for more non-peptide pulsed controls (maybe half the cells). with peptide or antigen if the target cells aren't expected to have the antigen already for this experiment. This can be done by adding peptide to a final concentration of 10 ug/mL and incubating at 1-4 hr for 37 C. Expose the target cells to as little DMSO as possible.
 * Ex calculation: 1000 ug/mL * y / (5 mL) = 10 ug/mL -> y = 0.05 mL (50 uL)
 * Make sure to pulse enough target cells. 4e6 target cells would be enough cells for 4 96 well plates since 1e4 target cells per well are used (4*96*1e4 = 3.84e6)
 * It may be best to incubate cells in a 14 mL or 5 mL round bottom tube rather than a pointed tube. It may be best to centrifuge in a pointed tube since it is easiest to separate the supernatant from the pellet in this type of tube. Note that it is not okay to incubate the target cells in a small tissue culture flask because the cells can adhere to the flask.
 * Note that for some experiments you may want to pulse some effector cells with peptide in the same way as a control. 1e6 cells per well are used sop it might be best to pulse 10e6 or 20e6
 * remember to start thawing FBS before adding chromium to target cells later


 * Wash away extra peptide by centrifuging <= 5x10^7 cells for 5 mins at 200Xg at room temperature. It may be best to do this spin with a pointed tube since it is easiest to remove the supernatant from the pellet in a pointed tube. Resuspend in 5 mL RPMI and centrifuge again. Discard the supernatant and resuspend in a volume of your choice (the original protocol mentioned 100 uL, but I think this may be too small of a volume). 1 to 5 mL of media may work.


 * Incubate the target cells with chromium. Add 20 uL FBS per 100 uL (I'm not sure how necessary this FBS step is) and 0.2mL 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 45 min for lymphocytes or 1-2 hr for tumor cells at 37 C, 5% CO2 incubator (try to avoid letting this incubation last longer than 1-2 hr). 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.
 * If any effector cells were pulsed with peptide for use as a control, then you might want to incubate these with chromium as well.


 * 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 appropriate volume of effector cells (or controls) and media (up to 0.1 mL total) 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.
 * Note that columns 1, 5, and 9 of a Lumaplate in our Topcount read slightly higher than the other columns. Although, a Lumaplate is not used at this step, one might want to arrange the cells in this plate the same way that they would be arranged later on when transferring the supernatent from these cells to the Lumaplate. Therefore, one may want to avoid using columns 1, 5, and 9.
 * “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)
 * How many targets should be expected? The number of targets in a fully confluent 175 cm^2 flask is about 1.75e7.
 * 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.
 * prepare controls that differ from test effector cells only in antigen specificity (nonsensitized cells or cells sensititized against irrelevant antigens)
 * 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.
 * Make sure to mix cells with triton well by pipetting up and down
 * Target cells not pulsed with peptide mixed with effector cells
 * Target cells pulsed with peptide mixed with effector cells that have not been "induced
 * Target cells alone (some peptide pulsed and some non peptide pulsed)
 * effector cells alone
 * effector cells and peptide alone
 * other possible controls:
 * Non-enriched T cells (naive and immunized)
 * Plain splenocytes with no 5 day induction (naive and immunized)
 * Example CTL Plate Map Portal 8-18-12


 * 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 3-6 hours 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.
 * Do not place the plate directly over the vent on the floor of the 55 C incubator in the radiation room. The plastic plate will melt if you do this.
 * 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)

Things to find out for protocol

Things found out for protocol >>
 * I should find out what type of scintillation fluid to use for a lumaplate for a chromium release assay.
 * Actually a Lumaplate does not require any scintillation fluid (http://www.perkinelmer.com/Catalog/Family/ID/LumaPlate)
 * Find out how to use Lumaplate. Do I dry the solution 1st? What do I use to seal it with again?
 * Basically just add the sample to the plate, let the sample dry, seal it with TopSeal-A and then count.
 * What will I be using to transfer the supernatent?I can use the Hand-E-Vac to take away the supernatent, but I can't use it to transfer the supernatent. Therefore to transfer the supernatent I think we will need to use a multichannel pipette.
 * Verify with Andrey that plates with radioactive material will be in centrifuge, and incubators.
 * He's aware of this.
 * find out how to pulse target cells from other protocol
 * Pulse target cells with peptide. This can be done by adding peptide to a final concentration of 10 ug/mL and incubating at 1-4 hr for 37 C
 * Ex calculation: 1000 ug/mL * y / (5 mL) = 10 ug/mL -> y = 0.05 mL (50 uL)
 * plastic shielding to line the inside of the lead sheilding (is this necessary?)
 * Raymon says to just use lead for everything. He said that shielding that looks like plastic is actually is a lead based plastic used for radioactive work. (see 7-16-12 e-mail for more details https://mail.google.com/mail/u/0/?ui=2&shva=1#inbox/13890f620ac4d9d6). He said the plastic shielding should be of the lead acrylic type. The shielding will be marked somewhere stating that it is lead.

Things to order

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