Hi.
I’m trying to make hybridomas from mouse spleenocytes and myeloma(SP2/0 and NSO), and some first attempts were successful. In experiments were many clones (approximately 8-10 in well) and It last during 1 year, then we stopped hybridoma making and after half a year decided to resume experiments. And unfortunately in fusions there was no fused cells at all, I saw some fibroblasts like cells and after 10 days the supernatant became yellow but still no clones. We suppoused that the problem is in HAT and PEG, and I made some fusions with different reagents(HAT supplements Gibco® HAT Supplement (50X) 21060017, new PEG with molar weights 4000 and 1500), but still no clones in more than 8 fusion. Also, we tried not to auctoclave PEG, as we did previously, but just passed through filter 0,45 um. But still no clones. The serum is the same for all fusions, and mice were no immunized in last attempts. The procedure that I followed is:
Mouse was killed by cervical dislocation, and using three pairs of tweezers and scissors the spleen was placed in iced free-serum DMEM.
Using scabrous surface of two slides the spleen was smashed. The solution of smashed spleen was passed through needle 18G three times, after 22G three times, and 26G three times. Then the solution was centrifugated at 100G, 5min and supernatant was discarded. The cells were resuspended in 10 ml of pre-heated 10%FBS-DMEM.
Myeloma (SP2/0 or NSO) was cultivated in 150 ml flasks, and detached from the surface of flask by Versen solution.
The cells were counted (I was trying not to count RBCs, but I noticed that there was too much RBCs; usually I get 20*10^6 spleenocytes from no immunized and 60-80*10^6 spleenocytes from immunized mouse) and mixed in ratio 2 spleenocytes : 1 myeloma cell. The mixture was centrifugated at 100G, 5min and cells were resuspended in 25ml of serum-free DMEM. Centrifugated again and supernatant was discarded.
The centrifugated cells were mixed by tapping the bottom of tube, and 50% PEG solution was added very slowly, approximately during 1-2 min (on every 100*10^6 cells I used 500 ul of PEG solution, usually I fused 30-40*10^6 cells and used 150-200 ul of PEG), during adding PEG the cells were mixed by shaking the tube. Than cells were placed in bathroom and incubated during 1 min. After the 25 ml of serum-free medium was added wery slowly, during about 5-8 min and during this time cells were mixed by shaking the tube, than the cells were centrifugated and resuspended in HAT medium (10% FBS-DMEM 500ml + 10ml of Gibco® HAT Supplement (50X) 21060017). The cells were placed in 24-well plates in concentration 2*10^6 cell in well, and placed in incubator.
And after several days I saw that supernatant became yellow, but no clones were observed.
So, I would be very glad for any advice for solution of this problem.
this will book will help
Antibodies: A Laboratory Manual 1st Edition (English, Paperback, Harlow, Ed Harlow David Lane, Edward Harlow)
I suggest you go for 1:10 ratio for fusion ......1: 2 is too low ......chance of getting fused cell is little bit difficult...................
Naveen Kumar B.T. , Thank you for sharig book and advice. I hope it will help.
Had a similar problem many years ago. Solved it by adding the following to media immediately before addition to fused cells (I add 1 % v/v glutamax from thermofisher + 1 % pyruvate from sigma + 50 uM mercaptoethanol to the medium containing 10 % FBS). Greatly improve hybrid colonies. Especially the glutamax appear essential. Add stock solutions on the day that you use medium as the glutamine and mercaptoethanol are unstable. Alternative is adding 10 % thymocyte conditioned medium - however not so easy to prepare.
Edmund Pool, Thank you so much for the answer. I will make expirement taking into consideration your advice and I'll write about results. I hope very much that it will finally work.
For fusion, you must be mixed splenocytes and myeloma cells in 10:1 ratio followed by centrifugation at 700xg for 10 mins. Then, add 1 ml of PEG-1500 per million cells very slowly @ 37C with gentle shaking. you may increase the time (up to 5 mins) and add DMEM medium (5 ml in 5 mins). seed the cells with 20% FBS medium on 96 well TC plate. Next day, add HAT medium to each well and left for selection. Media change should be done on or after 10 days post fusion and test culture filtrate on 13-14 days post fusion.
Hope it will solve the problem
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