Jacobs: Protocol Preparation of primary osteocytes and MDCK cells for immunostaining

Preparation of primary osteocytes and MDCK cells for immunostaining

JCC 2/24/11

Materials

• 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
• PBS
• Trypsin
• Trypan blue
• FBS (if necessary)
• Microfuge tube
• 15 ml centrifuge tube
• Pipettor/ pipet tips
• Pipet aid/ pipets
• Media with and without FBS
• Centrifuge
• Hemocytometer
• Kimwipes

Protocol

Collagen coating (can be done ahead of time and kept in refrigerator):

1. Dilute sterile collagen in previously filtered sterilized 0.02 M Acetic acid to final concentration of 0.15 mg/ml. To make 50 ml:
   1. Add 57 ul of glacial acetic acid to 50 ml DI water.
   2. Concentration of collagen can vary with batch- look on bottle.
   3. For 4 mg/ml concentration: Add 1.875 ml to 48 ml 0.02 M Acetic acid solution.
   4. Then sterilize entire solution.
2. This solution can be used approximately 6 times, and should be kept in refrigerator.
3. For each well of a 24-well dish, add 500 ul solution to each.
4. Coat for 1 h at RT.
5. Tilt to remove excess collagen and save.
6. If using immediately, rinse with PBS; otherwise dry the plates for 1 hour with lid off before storing at 4C.

Seed cells:

1. Wash cells with warm PBS.
2. Mix trypsin with pipet, because it settles.
3. Add 3 ml trypsin to each dish, and incubate for 3 minutes, or until cells have detached.
4. 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.
5. 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.
6. Spin on 1.5 for 5 min. Make sure to use a counterbalance.
7. Remove supernatant, and resuspend in 2 ml media.
8. Transfer 20 ul to microfuge tube.
9. Add 20 ul trypan blue to microfuge tube.
10. Add enough of solution (~20 ul, 10 ul each side) to hemocytometer. Clean with EtOH and kimwipes when done.
11. Count 4 areas and average. Multiply by 2 (for trypan blue dilution), then by 10,000 (volume) to give cells/ml.
12. Calculate the amount of media needed to add the correct number of cells to each well.
    1. For MDCK- 5k or 10k/well
    2. For primary osteocytes- 2k/well
13. Add media so that the total amount per well will be 0.5 ml.
14. Add cells.
15. Agitate dish a little to make sure that cells are evenly spread.
16. Check on confluency daily. Cells will settle towards the middle because it is slightly lower.
17. 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