Jacobs: Protocol Preparation of primary osteocytes and MDCK cells for immunostaining
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Preparation of primary osteocytes and MDCK cells for immunostaining
- 24-well glass-bottom dishes (MatTek P24G-1.5-13-F, have to be 1.5 thickness for confocal microscope)
- Collagen (Type 1, Rat Tail, BD 354236, no more than 2 months old)
- Acetic acid
- 24-well petri dish
- 100% EtOH
- Trypan blue
- FBS (if necessary)
- Microfuge tube
- 15 ml centrifuge tube
- Pipettor/ pipet tips
- Pipet aid/ pipets
- Media with and without FBS
Collagen coating (can be done ahead of time and kept in refrigerator):
- Dilute sterile collagen in previously filtered sterilized 0.02 M Acetic acid to final concentration of 0.15 mg/ml. To make 50 ml:
- Add 57 ul of glacial acetic acid to 50 ml DI water.
- Concentration of collagen can vary with batch- look on bottle.
- For 4 mg/ml concentration: Add 1.875 ml to 48 ml 0.02 M Acetic acid solution.
- Then sterilize entire solution.
- This solution can be used approximately 6 times, and should be kept in refrigerator.
- For each well of a 24-well dish, add 500 ul solution to each.
- Coat for 1 h at RT.
- Tilt to remove excess collagen and save.
- If using immediately, rinse with PBS; otherwise dry the plates for 1 hour with lid off before storing at 4C.
- Wash cells with warm PBS.
- Mix trypsin with pipet, because it settles.
- Add 3 ml trypsin to each dish, and incubate for 3 minutes, or until cells have detached.
- If large amount of cells, use old FBS instead of media to inactivate trypsin to avoid having a huge amount of media to spin down. Add 0.5 ml FBS or 5 ml media.
- Pick up cells in pipet and wash down at an angle to get any cells that are stuck. Transfer to 15 ml or 50 ml tube.
- Spin on 1.5 for 5 min. Make sure to use a counterbalance.
- Remove supernatant, and resuspend in 2 ml media.
- Transfer 20 ul to microfuge tube.
- Add 20 ul trypan blue to microfuge tube.
- Add enough of solution (~20 ul, 10 ul each side) to hemocytometer. Clean with EtOH and kimwipes when done.
- Count 4 areas and average. Multiply by 2 (for trypan blue dilution), then by 10,000 (volume) to give cells/ml.
- Calculate the amount of media needed to add the correct number of cells to each well.
- For MDCK- 5k or 10k/well
- For primary osteocytes- 2k/well
- Add media so that the total amount per well will be 0.5 ml.
- Add cells.
- Agitate dish a little to make sure that cells are evenly spread.
- Check on confluency daily. Cells will settle towards the middle because it is slightly lower.
- Once cells have reached confluency and for an area that is large enough to your liking, change media to 0.5% FBS to serum starve and leave for 2 days.
- History: JCC, last updated 2/24/11