AACF
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NIAID's Antimicrobial Acquisition and
Coordinating Facility AACF |
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-Biodefense Virus Panel |
SARS CoV (Corona Viruses) Assays
Hi/Lo Assay
(Southern Research Institute)
SARS Hi-Lo Assay Protocol NOTES · Cells are received in the morning, plated, and incubated overnight. · Batch size consists of 15 plates. 15 plates will be plated in the morning. · Drug additions are made to the cells the day after plating (4 plates at a time · All conditions must be kept sterile. · White clear-bottom plates are used for CellTiter Glo luminescence reads. · Black clear bottom plates are used for ATPlight luminescence reads.
REQUIRED MATERIALS/EQUIPMENT A. Equipment
B. Consumables
C. Materials
1. Reagents 1.1. P/S · Make 5.5 ml aliquots in sterilized eppie tubes, store at –20 C. · Yield: 20x 5ml aliquots of P/S
1.2 L-Glutamine
· Make 5.5 ml aliquots in sterilized eppie tubes, store at –20 C. · Yield: 20x 5ml aliquots of L-Glut
1.3 FBS
· FBS from Cell Biology should be heat inactivated for 30 min. · Make 50ml aliquots in sterilized eppie tubes, store at -20 C. · Yield: 10x 50ml aliquots of FBS
1.4 Media
Assay Medium: IMEM, 5%FBS, L-glut, antibiotics (w/o phenol red)
25ml Fetal Bovine Serum (Atlanta Biologicals Cat#S12450, Lot#B0053) Heat inactivated @ 56C for 30min. 5ml L-glutamine, 200mM [final conc. 2mM] (GibcoBRL Cat#25030-081, Lot31160728) 5ml Penicillin-streptomycin solution containing 1x104 U/ml penicillin and 1x104 ug/ml streptomycin [final conc. 100 U/ml penicillin and 100 ug/ml streptomycin] (GibcoBRL Cat#15140-122, lot#1174381) 500 ml DMEM; w/glutamine, w/o phenol red, gentamycin, proline (Cellgro; Cat#, Lot#)
1.5 CellTiter-Glo (See Promega for Complete Instructions) · Equilibrate Glo Buffer and Glo Substrate to room temperature prior to mixing. · Glo substrate is found in –20 C floor freezer · Glo buffer is found in the 4 C walk-in cooler · For the smaller Glo substrate bottles (size A), transfer 10 ml of Glo Buffer into the amber bottle containing the substrate. · For the larger Glo substrate bottles (size B), transfer 100 ml of Glo Buffer into the amber bottle containing the substrate. · Recap with rubber cap and mix gently, inverting ~10-20 times to dissolve all the substrate. · Transfer the solution into a sterile graduated cylinder.
1.6 1% DMSO in media (for validation; 1 drug plate is enough for 9 cell plates) · Add 19.8 ml of sterile dH2O into a 125 ml flask. · Add 0.2 ml of DMSO. · Pour into a sterile reservoir. · Aliquot 200 ul/well into a sterile 96 V bottom plate, columns 1-11. · In a separate sterile 96 well V bottom plate aliquot 200 ul/well into column 12 only.
Table 1: 96-well Z-plate format
Table 2: 96-well plate format
1.7 6688 (control drug) stock 30 mM in 100% DMSO
· Dilute 3.3 ul into 996.7 ul of complete media to get a 10X high dose of 100 uM in 0.33% DMSO. · Mix well.
2. Cell Receipt
2.1. The cell line for this assay is Vero 76 received from SRI’s Cell Biology department (Joyce Harwell x2239, x2004).
2.2. Warm assay media before going to Cell Biology Group by placing media in 370C Water bath in room KMII 607.
2.3. Immediately remove the cells from ice. Plate the cells as soon as possible. Do not allow cells to sit on the bench top longer than 30 minutes.
3. Plating Cells
3.1. Upon receipt, vortex cells for 10 sec to make a single cell suspension.
3.2. For a 96-well, plate with 50 ml per well, it requires roughly 5 ml/plate of cell suspension (50 ul/well x 96 wells). Make cell suspension up in sterile disposable Corning flask (either 125- or 250-mL, depending on volume needed).
To estimate the volume needed for cell plating: (5 ml/plate x # of plates) + 10 ml for tubing/priming = TOTAL VOLUME NEEDED
Example: If you are to plate 10 96-well plates: (5 x 10) + 10 = 60 (final volume of cells+media)
C1V1=C2V2
C1= 5000 cells/70 ul V1= 100 ml C2= 106 cells/ml V2= ?
2 ml
Add 2 ml of cell suspension to 98 ml of assay Media in a 250 ml sterile disposable flask.
3.3. Remove stir bar from flask of 70% EtOH with tweezers. Blot stir bar gently w/ sterile Kimwipe.
3.4. Place the sterile stirring magnet into the mixture of cells and media and place the flask on the plate stirrer. Allow contents of flask to stir for 5 minutes at speed 7 before plating.
3.5. Open pack of white, barcoded 384-well plates under the hood. Remove the total amount of plates you will be running.
3.5.1. Label the plates with the cell line and date.
3.6. Stack delidded plates on top of one another, with the A1 corner facing towards you on the lower right hand side. Remove the stacker on the far right side and place it over the stack of plates.
3.7. Gently grasp underneath the stacker unit and push the plates up further into the stacker to ensure plates are ‘locked’ in place.
3.8. Lift the stacker and place it in its position on the Multidrop, making sure that the A1 corner of the plates is facing you at all times to dispense cells into column 12.
3.9. The settings on the Multidrop under the hood in room KMII 607 should be at: § volume: 50 (to dispense 50 ul) § col (columns): 1 § Plate: 96
3.10. Ensure that the Multidrop’s power is turned on, the rotor cover is closed, and that the Multidrop is ready to dispense in 96-well format by pressing the “Prime/Drop” and then “Start” buttons on the left. If the unit does not dispense in the proper format, adjust the settings, restart the instrument, and double-check the dispense mode.
3.11. Transfer the flask of cells into the hood and stir for 5 min.
3.12. Under the hood, open a sterilization pouch containing a sterile cassette head with tubes for rows A (upper right) and H (lower Left) pulled from the plastic holder that goes into the media. This will make it possible to plate cells in rows B through G of column one. Pull apart the blue and white paper closest to the cassette dispenser head tips. Be sure not to touch the cassette tips to anything! Sterilization pouches can be found in the third drawer from the top underneath the drawers next to the sink in room KMII 607.
3.13. Do not completely remove the cassette tubing out of the pouch. Make sure the end of the tubing that will become submerged in the liquid does not touch anything.
3.14. Slide the cover over the Multidrop rotor towards the instrument, exposing the rotor wheels. Place the cassette under the rotor with the head to the right hand side. Be sure not to touch the tips. Fold the cassette over the grid in a clockwise direction. Make sure the tabs snap into place. Pull the rotor cover towards you. Place the tubing into the flask, ensuring that the tubing is fully submerged. Try not to block the hood’s air flow while manipulating the tubing.
3.15. Place the flask containing media and cells into the hood. Place the cassette tubing into the flask containing cells. Place the A and H tubes into a flask containing assay media and press the “Prime/drop” button.” Ensure that all the tips in the Multidrop cassette are dispensing liquid. Prime for about 5s, making sure all tips are dispensing equally.
3.16. Click ‘Start’ on the far right side (under the stackers) and the Multidrop will begin to dispense the 50ul of media into A12 and H12 and 50 ul of cells into B12 through G12 of the 96 well plates.
3.17. Once the Multidrop has finished adding media and cells to column 12 of all plates, lift the left stacker from the Multidrop, turn it so that the A1 well is facing away from you in the upper left hand corner, and place stacker in the right stacker slot.
3.18. Press the “Empty” button to return media back into the tube.
3.19. Clean the cassette head by priming it with 40 ml of dH2O followed by 40 ml of 100% EtOH.
3.20. Remove the cassette head and replace it with a sterile one with all the tubes attached.
3.21. The settings on the Multidrop under the hood in room KMII 607 should be at: § volume: 50 (to dispense 50ul) § col (columns): 11 § Plate: 96
3.22. Do not prime the tubing until just before you are ready to plate the cells!
3.23. Click ‘Start’ on the far right side (under the stackers) and the Multidrop will begin to dispense the 70ul of media into 11 columns of the 96-well plates.
3.24. Tilt the flask when the level becomes low to ensure that the tubing is totally submerged.
3.25. Once the Multidrop has finished adding cells to all plates, lift the left stacker from the Multidrop, and place within the black plate release unit. Plates will release from the stacker; lift the stacker up and over the plates.
3.26. Replace lids.
3.27. Place in 5% CO2 incubator, 37o C. Do not stack plates. Record plate barcode ID’s in the notebook.
3.28. Press the “Empty” button to return cells back into the tube. Clean the Multidrop tubing by running about 40 ml of dH2O then 40 ml ethanol through the cassette tubing. Remove the cassette head from the Multidrop so it can be autoclaved.
3.29. Record any problems/issues with the run in the notebook. Record the time and date that the cells were plated.
4. Control and Drug Addition 4.1. Drug Addition
4.1.1. See appendix 1 for Drug plate preparation. Be sure to notify compound prep manager at least 2 days in advance of what plates will be needed for the run.
4.1.2. For every 16 assay plates, create 1- 96-well V-bottom plate under the sterile hood that contains 200 ul/well of 1% DMSO in assay media into column 12 or 200 ul/well of appropriate control into column 12 (table 3).
4.1.2.1. To add control drug, obtain appropriate stock from -20 C (KMII 609)
4.1.2.2. Dilute 3.3 ml of the stock with 996.7 mL of sterile assay media.
4.1.2.3. Pippet assay media 1% DMSO and control drug in media into the appropriate wells in Column 12 (Table 3).
4.1.2.4. Cover plates with foil seals and label accordingly.
Table 3: 1% DMSO/Control Drug in column 12
4.1.3. Check FX trash in the hood. If full, empty tip boxes into regular trash and spent tips into blue barrel or red biohazard bag. Do not fill up red biohazard bag more than ˝ way with tips.
4.1.4. Under the FX hood in room KMII 610, press the “remote local” button on the keypad for the FX carousel.
4.1.5. Press either “Rotate right” or “Rotate left” to have the carousel deck labeled “C” facing you.
4.1.6. Remove lids and load 10 boxes of P250 tips (light blue bottoms, VWR catalog # BK717255) into deck “C”.
4.1.7. Rotate the carousel until the deck labeled “A” is facing you and place the cells test plates in this stacker.
4.1.8. Rotate the carousel until the deck labeled “B” is facing you and place the drug plates in this stacker.
4.1.9. Make sure that all foil seals and lids are removed from the plates. Leave lids under the hood on the table of the carousel.
4.1.10. Once all tips and plates have been loaded, press the “remote local” button.
4.1.11. On the FX computer, click on “Start”, then “Program”, then “SAGIAN Software” and “Biomek FX”.
4.1.12. To transfer drug to cell plates, open method “SAR add drug high dose or low dose.bmt”.
4.1.13. Place the control plate that you created into the P14 position on the deck (Click on the “Instrument Setup” line in the middle of the screen for proper positioning). Remove foil seal.
4.1.14. Click on the “Stacker Carousel Setup” line in the middle of the screen. Left click on the plates in deck A. For those plates that are called “drug”, make sure that the volume corresponds with the volume that is actually in the drug plate. Check all 4 drug plates to make sure the volume is correct. Click “OK”.
4.1.14.1. The Radio button next to “Sense the liquid level the first time…” should be checked for the drug plates.
4.1.15. Click on the green start arrow . Double check the setup, and if everything is correct, click “OK”.
4.1.16. Using the hand-held scanner (see Appendix 2 for instructions scan the drug and cell plates to associate which drug plate went to which cell plate.
4.1.17. Record time that drug was added on the plates and in the notebook.
4.1.18. After run has completed, replace lids for drug and cell plates. Remove the last box of tips from underneath Pod 1 and discard.
4.1.19. Repeat for next 4 plates until finished. Place plates back in 37o C 5% CO2 incubator in room KMII 607.
4.1.20. Empty spent tips from underneath FX when all runs have completed.
4 Virus Addition Notify BSL3 that we have plates ready for virus addition. When BSL3 staff are ready to pick up plates, bag them in small stacks and immediately take the plates to the BSL3 air lock. Virus will be added in a 25 ul volumn.
5 Detection of Cell Viability 5.2 Adding Glo (Luminescence) in BSL3 5.2.3 Preparing Glo solution
5.2.3.1 Count the number of plates you will be reading. Multiply that number by 12 ml. This is the amount of glo you will need.
5.2.3.2 Remove Promega Cell Glo reagent from –20oC freezer and the Glo reagent buffer from the 4oC and prepare. (Refer to section 1.5).
5.2.4 Open the Wallac 2100 manager.
5.2.5 Open Protocols.
5.2.5.1 Open Users
5.2.5.2 Open HTS Informatics
5.2.5.3 Select Promega Glo read
5.2.5.4 Click ‘Open’.
5.2.5.4.1 Check the settings:
5.1.16 Add 100 ul of glo reagent mix to each well and shack for 2 min at speed setting 5.
5.1.17 Send an email to the data person on when the run will be complete so they can get the data as well as filling out a data processing request form.
5.1.18 Record in the project’s notebook the order of the plates and any problems if they occur.
Appendix 1: Compound Preparation
Table 4: 1% DMSO in Columns 2-11
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