Hi, I'm Ashra Corona from the lab of Dr.Mitch Kronenberg in the Department of Developmental Immunology at La Jolla Institute for Allergy and Immunology. Today I'm going to show you how to prepare high five insect cells and infect them with VIR for the purpose of generating MA CD 1D te tremors. CD 1D teters are used by our lab and by other for the recognition of NKT cells.
VIR system are used widely to produce recombinant proteins including MS C class two TE tremors. So the procedure you will be shown has much wider applicability. This procedure involves initiating and growing up high five insect cells infect the cells with reovirus carrying CD 1D CDNA harvest supinate by centrifugation after four days of infection, lysis at concentration of protein in 150 millimeter sodium phosphate buffer, pH 7.4 purification of mouse CD 1D protein by nickel and T agros and iron exchange romme method concentration M CD one G protein to one me per ML using YM 30 concentrator where a enzymatic violation of mouse CD 1D by AVID D protocol, elimination of free BioTeam by S 200 column.
And finally, production of alpha gel CD 1D Tet. Though the whole procedure involves all these steps, but today we will only show you how to handle these cells and infect them with virtus for the purpose of generating more CD 1D tremors. Let's get started.
First of all, I would like to show you how to the TN five insect cells, the high five cell line was originated from the ovarian cells of the cabbage looper insect. They are used in this protocol because they are growing serum free, medium adaptable to suspension culture and produce high levels of recombinant protein at five ml of insect express medium in 25 centimeter square tissue culture F flas. No, that express five comes without glutamine.
So we need to add 10 ml of a hundred x glutamine pen strip, or glutamine alone into one liter of media. Now I am going to take the frozen vial of high five insect cells, thaw this in 37 degrees Celsius water bath immediately add high five insect cells to the flask and incubate for 30 minutes in 27 degrees Celsius. Incubator then aspirate the medium to get rid of the medium with DMSO gently without disturbing the cells attached on the bottom of the flask.
Now we need to add five ml of fresh medium into the flas after hawing, we now need to expand the cells prior to infection with vir. Monitor the cells every day when flask is nearly 70%confident. Bring the cells in suspension by banging the flask on both sides.
Transfer the cells to 1 75 centimeter square flask by adding 25 ml of insect express medium and five ml of cell culture split conference flask. One is to four or one is to five as needed. Do not let cells overgrow count the number of passages that you have split the cells.
Do not go over passage number 30 because it may cause aging of cells resulting in cell lysis. Cells need to be split every other day. If we split, one is to five.
When you reach the desired volume at the concentration of 1 million cells per ML infect cells, I generally grow up to two liter, which usually amount to 71 75 centimeter scale flask and approximately 25 ml per flask or four one liter ER flask and approximately 500 ml per flask. Growing cells up in ER flask is much faster at a hundred ml of culture and 400 ml of express five SFM medium and grow them in shaker at 1 25 RPM at 27 degrees Celsius temperature until we reach 1 million per ML cells of desired volume before infecting the cells, I would like to show you how to ti the belo virus and we will do it next. In order to tighter the virus, play 3000 to 5, 000 cells per well.
Well in a flat bottom 96 well plate in 200 microliter of in insect express five SFM medium. Let the cells settle at 27 degrees Celsius for at least 30 minutes. Make the serial dilution of avius stock.
One should do one is to tend dilution in one line of 96 value. Bottom plate using 250 microliter per well. Dilutions are made in express five SFM medium.
Using a multichannel pipet transfer a fixed amount, usually 20 microliter of different dilutions to the cells culture at 27 degrees Celsius Incubator for seven days to avoid the evaporation from the edge rows. Wrap the edges of plate with fil after seven days. Check the plate and determine which dilution of virus gives a 50%infection.
Then use a formula to compute the titer, which is expressed in PFU per ml. The formula is formed in most belo virus manuals. After ting, the virus, a required amount of virus to infect the cells for protein production, the multiplicity of infection should be between five to 10.
For example, if the titer of mouse CD 1D belo viruses a hundred million plaque forming units per ml and we have 20 million cells per flask, we need to add one ml to two ml of belo virus to to infect the cells. The cells should be infected for four days. On day four after infection, the cells should be detached, floating, swollen, and forming sausage like sheep.
Now it is time to recover the sup natant. Take the medium from infected flask transfer to 250 ml blue cap Falcon spinning bottles. They can be autoclaved and reused.
Fill the bottles evenly and sent to fuge the cells in so GSA rotor at 2000 RPM, 20 minutes at four degree Celsius. Collect the SUP natin and proceed for further purification. I have just shown you how to initiate, grow and infect the high five insect cells and titer the balo virus.
Using these cells, the most important aspects of this procedure is how to maintain the cells and how to know the exact titer of the baus. Once you have generated CD 1D tremors, they can be used as a powerful tool for analysis of glycolipid reactive T cells. This procedure for handling and ting the balo virus can be used for many other recombinant proteins as well.
So that's it. Thanks for watching and good luck with your experiments.
