1. It is best to pass the BOSC cells at 1:3-1:4 to prevent cell clumping
which occurs when the cells are passed at low density or when they are allowed
to become overconfluent. I pass the cells by rinsing once with PBS and then
trypsinizing for about 30 seconds. Note: Plating the cells may
be the most important step in obtaining high titers. It is extremely
important that the cells are not clumped and are at the correct density. Unlike
most adherent cell lines, the BOSC 23 cells do not form nice monolayers. Instead
they tend to clump before confluence (at which time the media will become acidic).
To overcome the clumping, I usually split the cells 1:1 one or two days before
I split them for transfection. This may need to be repeated if the cells do
not spread well. After the 1:1 splits, it is best to pass the cells 1:2 for
1 or 2 passages, and then at 1:3 or 1:4. The cells grow much slower than 293
cells, and the 1:3 split should take about 3-4 days to reach confluence. Transfections
are better if you plate the cells for transfection before the plate becomes
confluent.
2. Plate 2 x 106 cells/60 mm plate approx 18-24 hrs prior to transfection in 4 mls of 10% FCS without selection. Your transfection efficiency will be higher if the cells appear as single cells rather than clumps (see #1 on how to prevent this). Note: 2 x 106 cells/plate is not a typo. The dish should be about 80% confluent prior to transfection. It is also important to count the cells rather than estimating the split. The above cell number is optimized for MFG-lacZ. Other inserts may slow the growth of the cells and it may be necessary to plate more cells prior to transfection. I try to plate at a density so that the cells are 95-100% confluent at 24 hours after transfection
3. Just prior to transfection, change media to 4 mls of 10% FCS/DME containing 25 µM chloroquine.
4. Transfect by adding 6-10 µg DNA to CaCl2/H2O in a volume of 500 ul. Add 500 ul 2X HBS (pH 7.05) by bubbling. Immediately (within 1-2 minutes) add this solution to the cells. Note: It is also fine to halve all of the above reagants. Recipes for HBS and CaCl2 are on the attached pages. (It is probably fine to add the reagents without bubbling in the order suggested above. Add this solution within 1-2 minutes to the cells.)
5. At 10 hours, suck off the media and replace with 4 mls 10% FCS without selection. It is important that you do not leave the chloroquine longer than 12 hours. This will cause a large decrease in titer. The range for chloroquine treatment is 7-11 hours.
6. At about 36 hours posttransfection, suck off the media, add 3 ml of 3 x 105 /ml log phase growing B-cells and 3 ul of 4 mg/ml Polybrene.
7. After 8 to 12 hours of co-culture, carefully take out B-cells, spin down at 1000 rpm for 5 min and resuspend in 5 ml of fresh RPMI mediium.
8. Drug selection can be taken place at about 24 to 48 hour after the end of the coculture.