METHODS FOR BONE CELL CULTURE (osteoblast and osteoclast)

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Table of Contents
1.0 2.0 3.0 4.0 5.0 6.0 7.0 Osteoblast Isolation from Mouse Osteoclast isolation from Mouse Osteoclast isolation from Rabbit Procedure for generating mouse OB/OC co-culture Coculture fixation procedure Actinium/DAPI stainging Immunohistochemistry osteoclast protocol

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1.0

Osteoblast Isolation from Mouse Calvaria

Preparation of the enzyme solution (-20C) Collagenase 0.1% (+4C) Dispase 0.2% -mem 0%

0.10g 0.20g 100ml

0.05g 0.10g 50ml

wearing a face mask, weigh, mix and filter sterilize Preparation of the 10x Tripsin EDTA solution EDTA 0.155g Trypsin 0.50g NaCl 0.90g dH2O up to 100ml

First add the Trypsin to the most of the water, and adjust the pH with NaOH to 7.7 Then add the Trypsin ad the NaCl, then adjust the pH to between 7.4-7.6 Wearing a face mask, weigh, mix, filter sterilize and aliquot. Store at -20C Make 1x PBS-TC,(without Ca/Mg) from the 10x PBS by diluting (adjust the pH =7.2) 1. Cut the head off 1 day old mice and place in a petri dish with cold PBS 2. Isolate the calvaria from the head. (50-60 calvaria is good for seeding in 6 petri dishes) 3. Place the calvaria into a centrifuge tube and add 8ml of the enzyme solution 4. Place at 37C for 10 mins, in the shaking incubator at 200 cycles/min 5. Transfer the solution with a transfer pipette into a new tube, and place the tube on ice. This is fraction 1 which is not needed but saved till the end of the isolation. 6. Add a further 8-10 of the enzyme solution to the calvaria and place in the shaking incubator for another 10 minutes 7. Remove the enzyme solution into a new tube and keep on ice (this is fraction 2) 8. repeat steps 6 and 7 twice more, each time adding the fractions 3 and 4 to fraction 2. 9. Reapeat a final time using 9ml of the enzyme solution, and placing for 20 minutes in the shaking incubator. 10. This fraction 5 is also added to the previous fractions (fractions 2,3,&4). 11. Centrifuge at 4C 1500 rpm, 5 minutes 12. Remove the supernatant and resuspend the pellet. 13. Add 5 ml of the media (-mem with 10% FCS &1% P/S) and disperse, add 5ml further and again disperse 14. With the same media, fill to 48ml

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15. Using 6 petri dishes, add 8ml of the cells to each petri dish and add a further 2ml media. These volumes can be altered to ensure that there is enough for all the petri dishes that you have. 16. Incubate for 24hours 17. Change the medium (this will remove most other cell types that will be present contaminating the OB culture) 18. Incubate for 24 hours 19. Dilute the 10x trypsin with PBS to get a 1x trypsin solution ( you need about 2ml per petri dish) when splitting plan on making 4-5 new petri dishes for every existing petri dish. Ie split 1:5 if you are leaving over the weekend, or 1:4 if it is during the week. 20. Remove the media, add 5ml of warm PBS (37C), and wash gently and remove 21. Add 2ml of the 1x trypsin (37C) and place in the incubator till the cells begin to lift. Use a wide mouth pipette to collect the cells an place in the centrifuge tube 22. Add media to the plate using a plastic (wider mouth) pipette, wash the plate using a small volume each time, adding the solution to the centrifuge tube 23. The total volume of media after washing should be 10ml in the centrifuge tube 24. Add 5ml of cold media to the petri dish, up and down three times to wash, transfer this to the next petri dish and again up and down. Use this same solution to wash all petri dishes and then transfer to the centrifuge tube. (this cold media removes lifts any remaining cells) 25. Repeat point 24 again 26. Centrifuge at 4C 1500rpm for 5 mins 27. Repeat points 12 to 14 28. This time put 2ml of the cells into each petri dish, with 8mls of media. (this volume can be adjusted depending on the splitting ratio desired) 29. Incubate for 48 hours 30. The freezing media is made as 20% DMSO in -mem (with 0%) filter sterilize (make at 4C dont warm) 31. Repeat steps 20 to 26. Use 1 centrifuge tubes for ever 6 Petri dishes 32. Remove media, resuspend the dry pellet and disperse in 5ml of the 10% media. Combine all the tubes. 33. Count the cells: take approximately 50

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2.0

Osteoclast isolation from Mouse (Bone marrow cells)

Euthanise mice and isolate the 2 posterior feet, and add to sterile PBS or a-MEM using aseptic technique, Isolate femurs in a-MEM and flush out bone marrow using G23 hypodermic needle with 1 ml syringe in Petri dishes. Homogenize bone marrow and Collect into 50 ml falcon Centrifuge at 1500rpm, 4, 5 min. Discard supernatant and disperse pellet in 10% a-MEM (non heat inactivated). Remove incubated medium from OB and seed bone marrow cells on OB + Vit D and PGE2 Incubate at 90% RH, 5% CO2, 37c change medium after 2 days.
Time point fixation: day 4-5.

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3.0

Osteoclast isolation from Rabbit

Medium: Alpha MEM = 218 270 l, Protocol for 1 rabbit:

FCS hi 5% = 11500 l,

P/S 1% = 230 l

Prepare and set up under the laminar airflow chamber: 3x 50 ml-centrifuge tubes, one of those cut at 35 ml Box with ice 2x big Petri dishes: 1x with sterile PBS and 1x with Medium Plastic pipette Scissors, Scalpel and pinzette in alcohol 10 ml plastic pipette with a big hole (in order not to damage cells) Start the centrifuge at 600 rpm, 5 min, 4 C Put the legs of the rabbit in the Petri-dish, remove all muscles with the scalpel and scissors. Put in another Petri dish with sterile PBS. Under the laminar cut the clean bones into pieces 1. Take a 50ml tube already cut to 35 ml, put 6ml medium inside and add bones pieces. 2. Take the scissors out from alcohol and cut for 2 minutes the bone. 3. Add 7 ml 5% medium so that the cut legs are in 13 ml medium 4. Take supernatant &transfer with plastic transfer pipette in another 50ml tube on ice Point 2-4 you repeat 2 times. (total we cut 3 times) Add 13 ml of 5% medium, in 3 steps (5ml, 2x 4ml) to the bone, and transfer the medium with bones in a 50 ml centrifuge tube - Vortex 30 seconds Wait 2 minutes till bones settle, take the supernatant and add to the other supernatant on ice (now we had put 4 times the supernatant in the tube) Put the tube in the centrifuge: 5 minutes centrifuge 600 rpm, 4C Remove the supernatant and take the pellet

Important: dont use glass pipette for OC, but plastic pipettes with big opening in the tip.
Shake the centrifuge tube with the pellet. Add 5 ml medium and MIX very well Add 12 ml to have a final volume of 17 ml. (Add like this: 5 ml + 5 ml + 7 ml, and mix every time) Seed 300 l of cells in each well (48 well plate), or put 100 l Cell suspension on one glass, which lies on sterile Para-film in a Petri-dish 3 hours of incubation Remove the medium with the pump, carefully, put 500 l 5% medium back in the wells, remove the medium again Add 500 l 5% medium (with treatments when there is the need), or put the glass into the well-plate and add the medium. 48 hours incubation

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4.0

Procedure for generating co-culture

Seed at OB in a-MEM (heat inactivated) at least 6 hours before addition of OC. Remove incubated medium from OB and seed bone marrow cells freshly obtained as per OC isolation protocol, on OB + Vit D and PGE2 Incubate at 90% RH, 5% CO2, 37c Medium change after 2 days. Time point fixation: day 4-5

5.0

Osteoclast fixation

Wash with 1 ml 37C or room temperature PBS+Ca Mg to detach OB layer in CC or floating cells in OC culture. (500l for bone slices and 48 well plate) Fix once for 3 min with 1 ml (500l for bone slices and 48 well plate) of 3.7% Formaldyde at room temperature, the remove solution (this solution is prepared by diluting 37% Formaldehyde, 1:10 in PBS) Fix once for 10 min with 1 ml (500l for bone slices and 48 well plate) 3.7% Formaldyde at room temperature. Wash twice with 1 ml ((500l for bone slices and 48 well plate) PBS+Ca Mg at room temperature. Add 1 ml (500l for bone slices and 48 well plate) RT PBS+Ca for storage and add sodium azide for long conservation. Store at 4 deg (cold room).

6.0

Actinium/DAPI staining of fixed cells (English)

Preparation of Reagents: 5 l / cover slip (or per 2 disks) of Alexa Fluor 546 phalloidin (Nr.144) put in one Eppendorf- light protection!! (Put in a cupboard) and let the solvent evaporate under N2 gas) do up to 10 coverslips per time wrap the Eppendorf with aluminium foil When it has dried outadd 200l PBS+Ca+Mg/ into eppendorf and mix. DAPI-dilution: 200 l stock-solution with 10 ml PBS+Ca+Mg light protection (2 ml is used per coverslip or per 2-3 disks) Protocol:

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 Prepare a metal box with parafilm attached to the bottom with sticky tape. Put the cover slips/disks into hutches -covered with 3ml ice-cold acetone (propanone) 3 min (use Acetone only under the hood!!) Take the cover slips/disks out, drip dry and deposit them on parafilm Put 200 l of the dilution on each cover slip, 80ul on disks, add drop by drop to different areas of the coverslip, so that the whole coverslip is covered and none runs off. incubation for 30 min with light protection at room temperature 2x washing in PBS+Ca+Mg (fill the 6 well plate and tip upside down in the sink for coverslips- add and remove for disks) Put the cover slips into a 6 well-plate, or 2-3 disks per well put 2 ml DAPI-diluted solution on each well incubate 10 min in the dark at 37C 2-3x washing in PBS+Ca+Mg 2-3x washing in distilled water Label glass slides Put flurosave (tissue-teck) on the glass slide (for disks use the one in the small white bottle in the fridge) Tap the coverslip/disk edge gently on a paper towel and add the cover slip/disk on the glass slide, with cell side on the tissue tek.this means cell side down for coverglasses and cell side up for disks, and fix the borders with glue drying in the dark Storage at 4C in a folder wrapped with aluminium foil
7.0 Immunohistochistry osteoclast protocol

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Immunofluorescence for Osteoclasts

Take 2 cover slips per treatment and for controls in a 6 well plate, fixed with 3.7% formaldehyde in PBS. Control groups are always necessary, since osteoclasts normally have fluorescence. 1. Alcohol series; 40%, 70%, 80%, 90%, 100%, 90%, 80%, 70%, 40%. a. Put 2ml in each well of the 6 well plate. Swirl the solution briefly and then remove. b. This dehydration is necessary to make the cell wall permeable to the antibody. 2. Solution 1. 1ml per well a. Prewashing will improve the surface wettability. b. For approximately 24 coverslips you need 100ml PBS with Ca2+/Mg2+ c. Add 100mg 0.1% BSA kept in the fridge d. Add 50mg 0.05% Saponin (from sigma) kept in the chemical cupboard e. Dissolve for a long time on a magnetic stirrer. 3. Add 2ml of PBS with Ca2+/Mg2+ to each well, and wash 2-3 times 4. Solution 1 add 1ml to each well. Taking a break at this step is possible since the cells are fixed. 5. Put Coverslips on parafilm a. Add 100L of Solution 2 b. To prepare enough solution 2 for 12 coverslips, add 65L of NGS (5%) to 1235@ of solution 1. This makes a total of 1300L (ratio is 1:20) c. Nb. NGS is natural goat serum (Ziegenserum) kept in shelf 2 of the freezer at -20C. d. Stand for 30 minutes covered 6. Dry with kitchenpaper towel and put coverslips on new parafilm a. Add 100L of solution 3 (primary antibody) b. Stand for 2 hours covered. Instructions to prepare solution 3- primary antibody. Primary polyclonal antibody directed against phosphorylated tyrosine residues. Antiphosphorylated rabbit in 50% glycerin solution, since this has the glycerin then it will not be solidified even though it is in the freezer. It is kept in the electrophoresis room at -20 in shelf 1.

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For 12 coverslips mix in an eppendorf; 1,290L of solution 1 +65L of NGS (5%) + 26L of primary Antibody (3%) (the ratio is 1:50) total volume is 1300L, collect the contents of the eppendorf by a brief centrifugation. 7. Dry, and wash with solution 1, three times in a 6 well plate. 8. Put the coverslips on new parafilm a. add 100L of solution 4 b. wrap plate in Aluminium foil and stand for 1hr To make solution 4 the fluorescent antibody- rabbit (secondary antibody) the antibody is kept in the fridge. It is light sensitive and so the eppendorf should always be wrapped in Aluminium foil and stored in the dark. For 12 coverslips mix in an eppendorf; 1,290L of solution 1 +65L of NGS (5%) + 26L of secondary Antibody (3%) (the ratio is 1:50) total volume is 1300L, collect the contents of the eppendorf by a brief centrifugation. 9. Wash in the 6 well plate, wash three times with PBS followed by three times with water, do this by adding PBS to 3 consecutive wells of a 6 well plate, and Ultra pure water to the last three wells of the plate, wash by dipping the coverslips into each well of the plate. 10. Dry 11. Add 1 drop of the embedding solution for fluoresnce microscopy on a leveled slide. The coverslip should be layed so that there are no air bubbles. Note that the cell side of the coverslip should be down, touching the embedding solution. With glue go around the edge of the coverslip to seal it. Then the glue is left to dry. This is light sensitive and should be kept in the dark immediately. Can be kept in the fridge for approximately 2 months wrapped in Aluminum foil.

VOLUMES OF SOLUTIONS REQUIRED

Solution 1 For 6 coverslips 50ml of PBS with Ca/Mg 50mg 0.1% BSA 25mg 0.05% Saponin 617.5L Solution 1 32.5L NGS 604.5L Solution 1 32.5L NGS 13L primary AB For 8 coverslips 50ml of PBS with Ca/Mg 50mg 0.1% BSA 25mg 0.05% Saponin 855L Solution 1 45L NGS 837L Solution 1 45L NGS 18L primary AB For 10 coverslips 50ml of PBS with Ca/Mg 50mg 0.1% BSA 25mg 0.05% Saponin 1,045L Solution 1 55L NGS 1,023L Solution 1 55L NGS 22L primary AB

Solution 2 Solution 3

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Solution 4

604.5L Solution 1 32.5L NGS 13L secondary AB

837L Solution 1 45L NGS 18L secondary AB

1,023L Solution 1 55L NGS 22L secondary AB

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