Evaluating Natural Killer Cell Efficacy in Co-Culture Systems
School
Johns Hopkins University**We aren't endorsed by this school
Course
EN.580 580.412
Subject
Biology
Date
Dec 11, 2024
Pages
4
Uploaded by CaptainGull413
1 Co-Culture with Natural Killer Cell for Immune Targeting A subset of the immune system, natural killer cells, respond to activating signals indicating infection and remove the foreign or pathogenic cell (virus, bacteria, or cancer cell). Upon recognition, the natural killer cells cause holes in the foreign (or cancerous/infected host) cell membrane and then induce their death. The success and efficacy of the natural killer cells in removing foreign material is influenced by a variety of factors, including self vs. non-self identity and immune recognition as well as the relative abundance of each cell type. Understanding whether the NK cells are effective can be directly evaluated by assessing cell viability and death of the cells that they target. Goal: Interaction between co-cultured cells will be evaluated through cell health assessment via fluorescence-based labels. Materials 4T1 cells, mouse (source) NK-92 cells, human (source) RPMI1640 media (source) Myelocult H5100 media (source) IL-2 IS, 20 ng/μL(source)FBS, heat inactivated (source) 0.25% Trypsin-EDTA (source) PBS (source) 0.4% Trypan Blue stain (source) 100X Pam3CSK4, 1000 ng/mL (source), at -20 ˚C100X FSL-1, 10 ng/mL (source), at 4 ˚CC29, 1000 μM (source), at -20 ˚CMMG-11, 1000 μM (source), at -20 ˚CSparstolonin-B, 1000 μM (source), at -20 ˚C250X Hoechst 33452 (source), at -20 ˚CCalcein AM, 1 mM (source), at -20 ˚CEthD-1, 2 mM (source), at -20 ˚CComplete before class (be AS specific as possible): You will be assessing how your antagonist influences the innate immune response by combining effector cells, target cells, known stimulating molecule (IL-2) and your molecule. To stimulate the “killing” pathway you will need the NK-92 media supplemented with 500 mU/mL IL-2 after counting. Remember each condition should be plated in duplicate! Target Cell Identity: _____________________ Effector Cell Identity: ________________ NK-92 growth media recipe: ______________________________________________________________________ 4T1 media recipe: ______________________________________________________________________________ Antagonist concentration from Module 3: ___________
2 Control conditions: ______________________________________________________________________________________ Experimental conditions: _________________________________________________________________________________ _________________________________________________________________________________________________________ The ratio of effector:target cells is at least 5:1 and you should have concentrations validated for your molecule. The total volume in any well should be 100 μL. Consider the approach below, where we have completed two rows that create a set of required controls: target cells without effector cells or any molecules & target cells with effector cells and stimulation with IL-2. Target Cell # Effector Cell # Antagonist Agonist Volume of NK-92 Cell Suspension per well Volume of media + molecules per well Additional assay media volume 10,000 0 - - 0 μL0 μL100 μL10,000 50,000 - -50 μL0 μL50 μL10,000 10,000 10,000 10,000 10,000 10,000 We suggest making master mixes. Make sure you don’t forget to include any additional dilutions from combining cells, compounds, and media! Feel free to use the space below to plan your master mixes!
3 Day 1 Creating co-culture plates Today you will create the co-culture of the two cells and your pre-selected molecules. The instruction team created 96-well plates that have 10,000 cells/well of the target cells already plated. 1.Observe NK-92 flasks under the microscope to assess the morphology/appearance of the cells to check health. Take images of any noteworthy cells or conditions using the transmitted light filter, save the images on your USB. Description of Morphology: __________________________________________________________________________ 2.As NK-92 cells are grown as suspension, not attached to the dish, collect the entire volume of media with cells from their flask into a 15 mL conical tube. 3.Pipette up and down to dissociate any cell clusters and collect an aliquot of 10 μL of cell suspension. Centrifuge at 125 x g for 5 minutes. 4.Count cell aliquot with Countess II. Add 10 μL of cell + trypan blue solution to one side of a disposable chamber slide. 5.Insert one side of the slide into the automated cell counter (push until clicks into place). Allow autofocus to complete and select capture.NK-92 Density (Cresuspended): ___________________ cells/mL 6.Aspirate the supernatant off of the pelleted cells, be cautious as these cells are suspension growth and prone to accidental aspiration. Resuspend NK-92 cells to appropriate concentration in assay media, see Before class calculations.NK-92 needed (Ninitial): __________ per well # NK-92 wells: __________ Volume per well: __________ Total volume: _________ Volume media: ___________ Volume Cresuspended: _________ 7.Tilt the original (4T1) plate and use a micropipette or soft aspiration to remove the media on top of the 4T1 cells. BE CAREFUL NOT TO SCRAPE THE BOTTOM OF THE WELL & DAMAGE YOUR CELLS. 8.Gently add 100 μLof PBS to the side of the well. Tilt and remove PBS, again not scraping the bottom of the well. 9.Using a micropipette, add the appropriate solutions to each respective well of the 96-well plate. Remember not all cells will receive these cells, see Before class calculations. 10.Spin down the plate gently for 5 mins at 125 x g to settle the NK-92 cells onto the 4T1 cells. 11.Place 96-well plate with all created conditions into the 37°C incubator. Complete before class (be AS specific as possible): Calculate the necessary volumes of all materials needed to stain your entire plate. Remember to check stock concentration and desired concentration. All are added at the same time so a single master mix is needed. Total 4T1 media: _______ μL Hoechst: ________ μL1 μM Calcein-AM: ________ μL 4 μM EthD-1: _________ μL
4 Calcein-AM and Eth-D1 combine to provide a “live/dead” outcome in cells. You will want to check which label will identify live versus dead in addition to knowing how to visualize each. We will use these to identify the cytotoxicity that results from the co-culture assay. Calcein-AM excitation: _________ nm Calcein-AM emission: ________ nm EVOS Filter: _________ Eth-D1 excitation: _________ nm Eth-D1 emission: ________ nm EVOS Filter: _________ NOTE: The procedures for this day may require extended incubation, you are able to come up to 15 minutes early but cannot stay more than 15 minutes after class to accommodate other courses in the space. Lab Day 2 Co-culture and NK activation Today you will assess the impact of your various agonist, antagonist, and cell interactions within a key innate immunity response, perforin-dependent cell death of non-self targets. 1.Prepare LIVE/DEAD stain solution. Combine pre-warmed RPMI complete 4T1 growth media and add both dyes to the media according to the Before class calculations. Wrap mixture in foil. 2.Collect your experimental plate and in the BSC, tilt the plate toward you to GENTLY aspirate the supernatant. a.This step is key in removing many of the NK-92 cells. 3.Rinse each well gently by adding 200 μL of 37°C pre-warmed PBS solution to each well. Rotate/rock the plate by hand a few times to ensure effective rinsing. 4.Aspirate PBS to rinse away any remaining NK-92 cells. 5.Add 100 μL of Live/Dead stain in media to each well. Place in cell culture incubator for at least 15 mins (up to 30 mins). a.After 15 minutes, check the staining in a sample well to determine if more time is needed. 6.Remove plate from incubator. Gently tilt the plate and aspirate the staining solution. 7.Rinse wells by gently adding 100 μL of PBS and tilting to wash. Aspirate PBS and add fresh 4T1 media. 8.Using microscope, collect representative images of each condition in appropriate channels to assess living and dead cells. a.Consider 2 images per condition if time permits. 9.Quantify dye values from images by measuring signal intensity of individual channels. 10.One approach for this quantification is below: a.Within FiJi software (https://imagej.net/downloads) open all images from a single channel. b.Create a binary image with equivalent threshold values for each image (https://imagej.net/imaging/thresholding) c.Select Analyze>measure to quantify the total signal within each image.