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Revision as of 21:09, 18 September 2015
Bacterial Protocols | Mammalian Protocols | Day by Day |
- Mammalian Protocols
- We expanded a new line of HEK293FT mammalian cells from Divya Israni’s frozen cell stocks.
- We passaged our cells every 3-4 days to keep them alive, healthy, and viable for experimentation.
- We counted cells in order to prepare a certain concentration for plating.
- We plated our cells in 48-well tissue culture plates to grow our cells overnight in order for them to be optimal for transfections.
- We used an Epoch kit to Miniprep purified DNA plasmids for transfection.
- We prepared a mixture of plasmid DNA for transfection by diluting the DNA plasmids to equal concentrations and mixing them together.
- We used PEI-mediated transient transfection to get our plasmid DNA mixtures into the plated HEK293T mammalian cells.
- We let our transfection cell plates incubate overnight and an additional day.
- We transferred cells into 96-well plates ready for flow cytometry analysis.
- We finally performed flow cytometry using a BD Fortessa Flow Cytometer.
- Mammalian Cell Culturing
- Expanding a Cell Line from Frozen Stock
- Obtain a frozen stock of HEK293FT cells (ours came from our Mentor Divya Israni)
- Warm cell stock in 37 C bath
- Plate 0.5 mLs liquid cell stock
- Add 10 mLs media
- Incubate overnight
- Cell line is now ready, begin to passage and plate as necessary
- Splitting Protocols
- Basic Idea
- We need to keep the cells alive and well, so every 3 days you need to “passage” them into a new flask of a 1:10 dilution with new media and such. This is ONLY to keep them alive for later use such as plating and transfecting. Always make sure to work in a specific tissue culture room and to sterilize everything before entering the hood. This is to in general not contaminate the cells.
- Materials
- Flask with previous passage cells (or initial cell line if first passage) [referred to as flask 1]
- New empty flask [referred to as flask 2]
- DPBS (buffer to wash away excess media)
- DMEM (media for cells to utilize) w/ 25 mL bovine serum, 5 mL streptomyacin, 5 mL L-glutamine, 5 mL sodium something
- Trypsin (enzyme to lift cells from bottom of flask)
- Steps
- Ready waste container with bleach, put anything that touched cells into this
- Cells will be adhered to bottom of flask 1 containing media in 37 C, CO2 regulated incubator
- Flask should be vented (slightly open) when in incubator
- Under microscope cells should look like clouds and take up 70% of the space (this means they are 70% confluent)
- Tilt flask 1 so that media is away from bottom (with adhered cells) and remove media using seralogical pipette into the bleached waste bucket
- Pipette 4-5 mLs of DPBS into corner of flask 1 away from adhered cells
- This is to make sure that no cells are dislodged, which would occur if the reagent was pipetted directly onto the cells
- Rock flask back and forth to get media off of cells
- Make sure not to get any media in the neck of the tube
- Pipette 2.5 mLs of Trypsin into corner of flask 1 away from adhered cells (same motion as DPBS)
- Rock back and forth to get trypsin on cells
- Vent flask 1 and place in incubator for about 5 minutes (no more than 15 mins) for Trypsin to take effect
- Label flask 2 with Cell line, Passage number (first time will be “P0”, second “P1” etc), dilution (concentration of cells to media), initials, and today’s date
- FOR A 1:10 DILUTION (good for keeping alive, passaging every 3-4 days)
- Add 9 mLs of media to flask 2
- Make sure not to splash into neck and that all media is spread across the bottom of flask 2
- Look at flask 1 under microscope, should see cells moving in clumps
- Add 7.5 mLs of media to flask 1 (to bring total volume of flask 1 to 10 mL as we had already added 2.5 mLs of Trypsin)
- Tilt the flask and pipette up and down and across the bottom of the flask to wash the cells into the media (about 15 times)
- Try for no bubbles
- This will also break clumps, important for single cells to be passaged as they will get proper amount of media and grow better
- Put 1 mL of media+cells (for 1:10 dilution) from flask 1 into flask 2 that already has media
- Gently rock flask 2 forward and backward then back and forth, making sure not to get anything in neck
- Look at flask 2 under the microscope to make sure there are not too many clumps, if so continue to pipette mixture up and down
- Vent the flask and place in the incubator anytime the cells are not being worked on in the hood
- Your cells are passaged 1:10 and good for 3-4 days!
- FY1: If using for plates next day, passage 1:2 (5 mLs cells, 5 mLs media)
- Counting Cells Protocol
- You will need to count your cells before plating them, even before transfecting them.
- Want 50,000 cells/mL for density when plating
- Use 1 PCR per flask of cells
- If making many plates, mix all flasks together beforehand and ensure single cells only, then do following steps
- Aliquot some media using 1 mL pipette from flask 2 into the microtube
- Add 10 microliters trypan blue and 10 microliters cells+media to a PCR tube and let them sit for a minute - this stains the cells so they can be counted
- Take the half moon sheet for the Countess machine and add 10 microliters of the stain solution into the indented half moon
- OR simply pipette onto glass slide and count under microscope, want to finally know cells/mL
- Insert into Countess and make sure clean so that there is no fuzziness or it will be counted as dead cells
- Want at least 90% of cells to be live
- Good number is over 1x10^6
- Plating Cells Protocol
- Get sterile, tissue cultured 48 well plate
- Going to put 250 microliters into each well so total necessary media is 12.5 mLs (round up to 15 for safety)
- Math: (50,000 cells/mL)(15 mL) = (live cells/mL from Countess)(X mL) and solve for X
- Pipette X mLs of media into reservoir and pipette 15-X mLs of media also into the reservoir
- Make sure to mix thoroughly and frequently as to ensure only single cells are plated
- Eppendorf large Multichannel settings:(at least 1000 ul multichannel pipette)
- Dispense
- 250 microliters
- Speed 4-5
- 4 times drop
- tip x x tip x x tip x x tip x x
- Look at plate under microscope to make sure that all wells look about the same and that it looks like 50,000 cells/mL
- Gently tap plate on all four sides against wall to make sure cells spread out
- Incubate at 37 C, CO2 regulated
- Transfection Protocol
- DNA Mixture
- Have spreadsheet made in advance about which plasmid goes into which tube
- 8 strip PCR tube holds all of DNA + NACL + PEI
- Each PCR tube holds enough for 8 wells = 200 microliters
- will only be using enough for 6 wells = 150 microliters to run positive and negatives in triplicate
- which is 25 microliters/well
- Each tube should get 4 plasmids of 50 ng/ul
- This can be adjusted depending on how many plasmids need to transfected, important number is that each well gets 250-300 ng/ul DNA
- If tube only needs less than 4 plasmids must use blank plasmid (ps-21)
- for example: full integrase control has transfection marker, reporter, full integrase and blank plasmid
- for example: dark cell control has blank plasmid, blank plasmid, blank plasmid, blank plasmid
- All DNA should be diluted to 50 ng/microliter in advance
- Or less if using more plasmids
- Add 10 microliters of each 50 ng/microliter plasmid to tube for 4 plasmids total = 2000 ng DNA total
- Should have 40 microliters of DNA in each tube
- GO TO TC HOOD
- Add 60 microliters .15 M NaCl to dilute DNA and bring total volume to 100 microliters
- Mix NaCl + PEI to be 42:8 for all tubes
- Each tube gets 50 microliters of this mixture, add extra wells to ensure enough
- Mix thoroughly in reservoir
- Add 100 ul NaCl + PEI to each tube
- Mix thoroughly!! PEI is what mediates transfection
- Let sit for 15-20 mins
- Use large yellow integra pipette (at least 125 ul multichannel pipette)
- aspirate 75 microliters
- will dispense 25 microliters three different times (into three rows of the negative 48 well plate)
- Same protocol for positive plate
- Incubate overnight
- Incubate all of next day
- Transferring from 48 to 96 Well Plate
- Tilt plate so that media is aggregated, aspirate 250 microliters (all) of media from 48 well plate using large eppendorf multichannel pipette
- Add 50 ul of trypsin to each well (so for 48 well plate need at least 2.5 mL of trypsin in reservoir)
- MAKE SURE to move plate north to south and east to west and tip back and forth to make sure that trypsin gets ALL cells off of bottom of plate
- Incubate for a 10 mins and make sure to look under the microscope - should see single spheres that move if you tip the plate
- DO NOT want cells to come off in sheets, will mean too many cells and likely poor transfection
- Add 50 microliters of media to each well (again at least 2.5 mL of media in reservoir)
- Use yellow integra multichannel pipette
- Mix many times
- Aspirate 100 ul and transfer to 96 well plate
- Flow cytometry or fluorescence microscopy
- Cleaning Up
- Pipette bleach into anything with old passages of cells in it (will kill them)
- Pipette that into waste container already containing bleach (let sit for about 10 mins) then wash down drain while running water
- Flask shut tightly and reservoir, both thoroughly cleaned into biohazardous waste
- Wipe down inside of hood with ethanol
- Close hood and leave UV light on for 15 mins to sterilize
- General Tips
- 70% ethanol on ANYTHING entering hood, especially gloves
- Do not push window above the sash level (dictates how much air gets in)
- Try to keep things in the hood and not take too much stuff in there
- Always keep incubator at 37 C and 5% CO2
- Media colors: pink = oxygenated; red = grown; orange - yellow = overgrown
- Confluence = how close together the cells are; want 70% when passaging or plating
- HEK203T cells are adhesive so do not move flask too much
- When in the hood do not pass anything over (eg hand) open containers of cells
- Labelling new flask for passage: (Lab name) (Cell line)
- (passage #) (day of the week) (dilution)
- (date) (initials)
To transfect our sets of plasmids into mammalian cells and assay for fluorescence using flow cytometry, we used the following protocols in our experimental pipeline.