I need someone to help me with each step assuming that I have 20ml's of adipose tissue. There's nothing wrong with this protocol, I'm just really bad at filling them in and want to get this right.
1. Dissect vessels and visible connective tissue from the adipose tissue, cut it in small pieces (3 to 4 mm)
and fill into Falcon tubes.
2. Prepare the Collagenase Working Solution (CWS):
• Determine the approximate volume of the tissue (A ml).
• Calculate the Units of Collagenase NB 6 you need:
• Final volume: 2 x A ml.
• Final concentration 0.3 U/ml (specific Collagenase activity see Certificate of Analysis).
• Dissolve Collagenase NB 6 in A ml of PBS.
• Add CWS to the adipose tissue into the Falcon tube.
3. Incubate at 37 °C for 30 to 60 min in a water bath with constant gentle agitation (Temperature should not
vary more than +/-1 degree as adipocytes are very sensitive to temperature fluctuations).
4. Stop digestion by adding 2 vol of DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
5. Filter through a 120 µm mesh.
6. Incubate the filtrate for 5 min at RT:
Upper phase: adult adipocytes + oil, lower phase: stromal-vascular cells including pre-adipocytes.
7. Centrifuge at 400 x g for 10 min using a 50 ml centrifugation tube.
8. Discard supernatant.
9. Dissolve cell pellet in ELB.
10. Incubate at RT for 10 min with gentle agitation.
11. Centrifuge at 400 x g for 10 min.
12. Discard supernatant.
13. Resuspended in DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
14. Filter through a 100 µm mesh.
15. Wash 2 times with DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
16. Stain the cells with Trypan blue.
17. Count the cells using a hemacytometer.
So from step one,
1. Dissect visible connective tissue and vessels of the 20ml's of adipose tissue.
2. Prepare the Collagenase Working Solution (CWS):
• Determine the approximate volume of the tissue (20 ml).
• Calculate the Units of Collagenase NB 6 you need:
• Final volume: 2 x 20 ml.
• Final concentration 0.3 U/ml (specific Collagenase activity see Certificate of Analysis).
? So how much collagenase is used and how much PBS should it be dissolved in? I'm guessing 40ml's of PBS and 12mg's of collagenase? So I'll be using several 50ml falcon tubes? Can you tell me how much of everything to put in each tube so that it's as clean as possible?
3. Incubate at 37 °C for 30 to 60 min in a water bath with constant gentle agitation (Temperature should not
vary more than +/-1 degree as adipocytes are very sensitive to temperature fluctuations).
4. Stop digestion by adding 2 vol of DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
? So the total volume of everything in each falcon tube should be multiplied by two and that's how much DMEM to add? So I'm going to really need many tubes?
5. Filter through a 120 µm mesh.
6. Incubate the filtrate for 5 min at RT:
Upper phase: adult adipocytes + oil, lower phase: stromal-vascular cells including pre-adipocytes.
7. Centrifuge at 400 x g for 10 min using a 50 ml centrifugation tube.
? Again, how many tubes given the 20ml tissue variable?
8. Discard supernatant.
9. Dissolve cell pellet in ELB.
10. Incubate at RT for 10 min with gentle agitation.
11. Centrifuge at 400 x g for 10 min.
? Just estimate how many tubes I'll need for this.
12. Discard supernatant.
13. Resuspended in DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
? How much?
14. Filter through a 100 µm mesh.
15. Wash 2 times with DMEM/F12 (completed with FBS, Penicillin-Streptomycin, Amphotericin).
? Very basic, but wash the cells with PBS right? How much should be used each time?
So I think you need to have 20 ml of PBS containing 6 Units of collagenase. I don't remember the units per mg of the collagenase. Just divide 6 Units by the units per mg on the certificate of analysis and add that amount of mg. It seems that you take the 20 ml of PBS containing collagenase and add to the 20 ml of tissue for 40 ml final. Then if you add 2 volumes of the DMEM/FBS, etc to stop, that would be adding 80 ml for a total volume of 120 ml or 3 falcon tubes. So for ease, I would do the digestion in 1, 50 ml falcon tube, and then divide into 3 tubes, ie 13.3 ml each. Then add the stop solution, ie the DMEM/F12, 26.7 ml to each of the three tubes. Once you pass through the mesh, you will have a smaller volume, and less tubes. It may fit into 2 tubes or less.
I have no idea what volume to resuspend the cells in the ELB or the DMEM/F12. I would use as much that you can, and that will fit in the tube. I think the more you wash, the cleaner the cells will be. What matters is your final resuspension. You may want the cell s per ml at a certain number.
Do you want the adipocytes or the pre adipocytes? I think the adipocytes would be in the supernatant, according to your protocol and you may want to keep the wash volumes low. Can you pellet adipocytes after you isolate them? You may need to ask someone who has used adipocytes before what to do.
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