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:
 * 2) Add 57 ul of glacial acetic acid to 50 ml DI water.
 * 3) Concentration of collagen can vary with batch- look on bottle.
 * 4) For 4 mg/ml concentration: Add 1.875 ml to 48 ml 0.02 M Acetic acid solution.
 * 5) Then sterilize entire solution.
 * 6) This solution can be used approximately 6 times, and should be kept in refrigerator.
 * 7) For each well of a 24-well dish, add 500 ul solution to each.
 * 8) Coat for 1 h at RT.
 * 9) Tilt to remove excess collagen and save.
 * 10) 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.
 * 13) For MDCK- 5k or 10k/well
 * 14) For primary osteocytes- 2k/well
 * 15) Add media so that the total amount per well will be 0.5 ml.
 * 16) Add cells.
 * 17) Agitate dish a little to make sure that cells are evenly spread.
 * 18) Check on confluency daily.  Cells will settle towards the middle because it is slightly lower.
 * 19) 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