Induction+of+Cytolytic+Activity+in+CTL+Precursors


 * __ Induction of Cytolytic Activity in CTL Precursors __**

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"

Forms and Templates http://stanxterm.aecom.yu.edu/wiki/index.php?page=Forms_and_Templates

>> 5%, 10%, or 20% FBS, heat-inactivated 1 hr at 56°C >> 1% nonessential amino acids >> 2 mM L-glutamine >> 2-BME (Beta Mercaptoethanol) >> 100 U/ml penicillin >> 100 μg/ml streptomycin sulfate >> for up to a week. If a precipitate forms, a fresh solution >> should be prepared. The precipitated solution has >> proven to be toxic to cells. Stock solutions should also >> be stored in the dark since it is readily decomposed by >> light.
 * //Materials://**
 * Effector cells (aka. responder cells, T-cells, lymphocytes, spleenocytes)
 * Complete DMEM medium. Made up of complete DMEM medium containing
 * Dulbecco’s modified Eagle medium, high-glucose formulation, containing:
 * Sensitization DMEM Medium
 * Complete medium
 * 1 mM sodium pyruvate
 * Target cells (aka. stimulator cells, the tumor cells we grow)
 * mitomycin C in HBSS
 * Should use 0.5 mg/mL solution.
 * Generally, solutions at pH 6–9 can be stored at 0–5 °C
 * A 0.5% solution in water has a pH of 6.0–7.5.9. A saturated solution has a pH of 5.0–9.0.3
 * http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/m4287pis.Par.0001.File.tmp/m4287pis.pdf
 * Con A or IL-2 (optional adjuvents)
 * 15mL conical tubes
 * Centrifuge (will centrifuge at 1000rpm)
 * 24-well flat bottom microtiter plate, 2mL capacity with lid

//**Procedure:**//


 * Note about this secondary bulk culture to induce more effector activity.
 * There are many situations in which you would not want to do this secondary bulk culture. The bulk culture will expand the number of T cells you have. You may obtain so many T cells that they overwhelm and kill of the target cells with every sample type. In this situation, you would be more likely to see killing, but less likely to discern differences between T cell populations from different mice. If you want to see differences in T cell population, you may want to use the T cells right away for the killing assay and skip the secondary bulk culture. Of course you would only want to do this if it is still possible to have a high enough killing for detection.
 * Add mitomycin C to target cell suspension to 25ug/mL final concentration.Incubate for 20 minutes in a conical tube tightly capped protected from light in a humidified 37C, 5% CO2 incubator.
 * Treat as many target cells as you need to have a 1:1 or 2:1 ratio of E:T when all or half of your effectors from the spleen are used. You will want to use as many splenocytes as possible so you have enough cells later on. You may want to split the splenocytes in half so that you have half for a 5 day induction and half that can be tested immediately.
 * Ex. Calculation: 1000 ug/mL*y/(3*mL) = 25 ug/mL -> y = 75 uL of 1 mg/mL mitomycin C in 3 mL media
 * Mitomycin C blocks cell divison
 * Fill tube containing target cells with complete DMEM and centrifuge 5 minutes at 1000 rpm at room temperature. Discard supernatant and wash 3 times with 14mL complete medium using these centrifugation conditions, ending regime with target cell pellets.
 * 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
 * Note that the word "pulse" is used because the cells are just briefly exposed to the peptide.
 * Remove media with peptide from target cells and wash cells with clean media 2X.
 * Using sensitization medium, adjust effector cells to 2-10x10^6 cells/mL and target cells 1-6x10^6 cells/mL.
 * Mix targets and effectors
 * Optimal concentrations of target and effector cells will vary somewhat with the nature of the antigenic differences and with prior activation of cells.
 * May want to try several E:T ratios
 * 1:1 (we'll just use maximum number of splenocyte or T cells that we have (or 1/2 instead of all) and mix them 1:1 with target cells)
 * 2:1 (maybe 4*10^6 E to 2*10^6 T)
 * Add 1mL each responder and stimulator cells to the wells of a 24-well microtiter plate. Final cell concentration shouldn't exceed 12x10^6 cells/well in a volume of 2mL.
 * Alternatively use a flask instead of wells. So for example 10 million targets and 10 million effector in about 12-15 mL in a T75 flask.
 * Culture cells 5 days in a humidified 37C, 5% CO2 incubator. Leave lids attached but allow air exchange.
 * Some fresh media containing recombinant human IL2 (use human even for mouse cells since it actually has a stronger effect) should be added on 1 day after the culture is started and 3 days after the culture is started. On the 1 day after addition use about 50 U/mL IL2 and on day 3 use about 100 U/mL. IL-2 will enhance proliferation.
 * Note that some people add ConA. However, IL-2 is the more modern way of inducing proliferation since the purpose of ConA is just to get the cells to start producing IL-2. Producing IL-2 itself used to be too hard for scientists to do.
 * Example Calculation: 50*U/mL*1*mg/(4*10^6*U)*1*mL/(0.1*mg)*20*mL = 0.0025 mL = 2.5 uL
 * So 2.5 uL of 100 ug/mL IL-2 would be required for a 20 mL culture
 * Harvest effector cells. May need to use a cell scraper to detach adherent cells if desired.
 * Using a disposable pipet, transfer cells to a 15mL conical tube
 * Centrifuge for 5 minutes at 200 x g at room temperature.
 * Resuspend pellet in complete DMEM at 1-5x10^6 cells/mL in a tightly capped tube.
 * Maintain at room temperature or ice until CTL activity is assayed by 51Cr release or other methods.
 * May want to use T cell enrichment kit.
 * May want to use T cell enrichment kit.