3T3 Cells Preparation for Staining Protocol
Cells
We use 3T3 Fibroblast cells.Fibroblasts are cells of mesodermal origin that make the structural fibers and ground substance of connective tissues.
They are characterized by an abundant and branched cytoplasm surrounding an elliptical, speckled nucleus having one or two nucleoli.
Fibroblasts make collagens, reticular and elastic fibers, glycosaminoglycans and glycoproteins found in the extracellular matrix or ground substance.
Medium
- 430 ml DME high Glucose (Irvine Scientific #9031 - $13.89/500ml)
- 50 ml Fetal Bovine Serum (Irvine Scientific #3000 - $64.96/100ml)
- 5 ml antibiotic: Penicillin-Streptomycin (Irvine Scientific #9366 - $14.02/100ml)
- 5 ml HEPES buffer 1M (Irvine Scientific #9319 - $38.84/100ml)
- 5ml L-Glutamine (Irvine Scientific # 9317 - $18.29/100ml)
- 5ml MEM Non Essential Amino Acids solution 10mM (100X) (GibcoBRL #1114-050 - $12.20/100ml)
Cell preparation
- Get the cells out of the -80ºC freezer. Thawing cells should be fast: put the tube directly in a 37ºC water bath.
- Add 1 ml medium and pipette medium + cells into a culture tube.
- Add 12 ml medium and gently pipette up and down.
- Spin 5 minutes at 1000 rpm.
- Aspirate the medium (be careful not to aspirate the cells).
- Flick the tube and add 10 ml of medium and gently pipette up and down.
- Put those 10 ml in a tissue culture dish (10 cm in diameter) (or just a part of those 10 ml if you want a more diluted concentration of cells, and then complete with fresh medium up to 10 ml).
- Put the tissue culture dish in a CO2 incubator at 37ºC for ~ 1 day. Check the % in confluency after 12 hours.
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