Fusion Protocol:
4mol H1 --->1 mol He4
4(1.008)--->4.003
4.032--->4.003
=.029g/mol mass loss
E=mc2
=.029g(3X1010cm/s2)2
=.029g(9X1020cm2/s2)
=.261X1020cm2/s2
=26,100,000,000,000,000,000 Ergs/mol
-or=500,000,000 kcal/mol
Alternative Fusion Protocol:
(Derived from Harlow Manual via Lees Lab 11/16/98)
-Final Boost mice ~4 days prior-iv/retro-orbital or tail vein
-Make media w/ and w/out serum (medias 1 and 2, below)
-Sterilize apparatus, make sure ample supply of plates, tips, etc.
-Night before Fusions, dilute Sp2 myeloma cells ~1:3 into
media 3 at ~5X105cells/ml (5X106/plate)
-Day of fusion, sac mouse, exanguinating by heart puncture (25 gauge needle)
-Remove Spleen, trims fatty tissue as much as possible, and
wash whole spleen 3 times in media w/out IFS
-release spleen cells by washing and straining
-Do in ~10ml media w/out serum
-remove to 40 ml conical tube, leaving large debris
behind
-Let large tissue settle in tube 2 minutes, and take off
sup. to new 40 ml tube
-Add media w/out serum to 40 ml
-Wash spleenocytes twice in serum-free media 1000rpm, 5 min
-Second wash, also wash Sp2 cells once with media w/out
seurm. Have enough cells for needed fusions
-Bring cells up in media w/out serum. Count cells
-Combine Sp2 to spleenocytes at a ratio of 1:3-5 in 50 ml
tube. Bring media up to 40 ml total. 1000rpm 5 min
-Aspirate carefully and completely
-Tap tube gently at the base
-Add 50% w/v PEG 3,700, 1ml in 1ml pipet slowly over 1 min
-Stir consistently without crushing
-Fill a 10 ml pipet with media (no serum) and add 1ml media
over the next minute with stirring
-Add final 9 ml over the next 2 min, with stirring
-Centrifure the cells at 800 rpm for 6 min in 40 ml tube
-Remove sup gently and resuspend cells in 10 ml media 1
-Transfer to 200 ml of media 1 in sterile glass bottle
-Plate out in 2000 wells, 100 microliters each
-Feed 4 days after fusion with further 100 microliters
Media 1
Media 2
*
DME
DME
20%IFS
P/S/F
P/S/F
L-gln
L-gln
1XOPI (Sigma O 5003)
1XHA (Sigma A 9666)
10%HCF(Fisher/Origin 50-0615)
*RPMI is recommended, DME works
Media 3
DME
10%IFS
P/S
L-gln