G418 (Geneticin): toxic: may cause birth defects; harmfuly by inhalation, in contact with skin and if swallowed. An antibiotic active against bacteria, yeasts, plant and animal cells.
DIMETHYLSULFOXIDE (DMSO): combustible, causes irritation, harmful if swallowed; keep away from eyes, skin, clothing; keep in a tightly sealed container. Inhalation may cause nasuea.
LIQUID NITROGEN: liquid form can cause severe burns. Avoid contact
lab coat
gloves (when measuring G418 and DMSO)
insulating gloves when going into the LN2 tank
Plastic disposables used for tissue culture of NON-HUMAN cells can be placed in the regular trash.
Drain all solutions from flasks before putting into waste.
When flasks in hoods are filled with liquid waste, dispose of contents down the drain with water running, rinse flask, add enough bleach to cover the bottom, and put back in place.
Pasteur pipets DO NOT go in trash. Put them in sharps container next to each hood.
Wipe down hood before and after use with 70% Ethanol (spray bottle).
DonÕt leave paper towels, spills, extra equipment, etc. in the hood.
Wash hemacytometer immediately after use with ethanol and dry.
RMA-S, EL4, E.G7
Monday, Wednesday:
1/5 = 10 ml fresh media and 2.5 ml cells
1/20 = 7.5 ml fresh media and 2.5 ml from 1/4 dilution
Friday:
1/10 = 9 ml fresh media and 1 ml cells
1/100 = 9 ml fresh media and 1 ml from 1/10 dilution
B3, GA4
Every 7-10 days:
Count CTLÕs and suspend 1.8 x 105 cells in 6 ml CTL media. Then aliquot 1.5 ml of cells in first 4 wells. Pass .5 ml into next well containing 1 ml fresh media.
3x104/well 104/well 3x103/well
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1 ml .5---} |
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1 ml .5---} |
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á Stimulator Cells:
5 x 106 E.G7 cells irradiated with 20,000 rads
Wash 3X in RP-10 (10 ml each)
á Feeder Cells:
C57BL/6 spleen irradiated at 2000 rads
1. Aliquot 5 ml of HBSS to 3 small petri dishes.
2. Flame a screen and place in one of the dishes.
3. Wash spleen 1X in first dish.
4. Place spleen on screen in second dish.
5. Grind spleen with plunger of 3cc syringe.
6. Wash second dish with HBSS from third dish.
7. Wash 2X in HBSS (10 ml each).
Pool stimulator and feeder cells in 25 ml of CTL media and aliquot 1 ml per
well on 24 well plate.
to freeze:
1. Thaw a tube of cell freezing media (CFM) (which is 10% DMSO in FCS).
2. Count cells, spin.
3. While cells are spinning, label Nunc poylpro freezing vials.
4. Resuspend cells at 2-10 x106 cells/ ml in CFM, aliquot 1ml per tube.
5. Place tubes in a sytrofoam rack, place rack in -70 freezer overnight.
6. Next day, transfer cells to the LN2 tank. Wear insulating gloves, work quickly, and be sure to record your placement in the LN2 book!
to thaw:
1. In 15 ml tube, place 4 ml appropriate media.
2. Bring vial of cells out of tank. Place in a 37o waterbath JUST until liquid.
3. Add thawed cells into media.
4. Wash freezing vial with media from tube.
5. Spin 5 minutes at 1000 rpm.
Vacuum off supernatant and raise to appropriate volume.