Preparation of Isolated Adipocytes/Pre-adipocytes from Rats

The following protocol outlines the procedures for obtaining adipocytes and pre-adipocytes from the epididymal fat pads of rats. It has been optimized for obtaining primary differentiated adipocytes, however, it has been used successfully to isolate pre-adipocytes also. The procedures are aimed at obtaining fully metabolically active adipocytes that will respond to insulin with an ED₅₀ less than 100 pM in the lipogenesis assay. Other protocols which are primarily aimed at obtaining preadipocytes may use different collagenase concentrations, digestion times and washes. Where applicable notes have been included that may increase the numbers of pre-adipocytes obtained if the primary adipocytes are not needed.

Reference: Protocol from the University of Lund, Version 860415

A. Supplies:

1. Animals: Rats should be 36+/-1 days and 120-150 g at the time of experiment for best results. Experiments should be performed on animals that have ‘rested’ after receipt in the facility.

2. Dissecting instruments including 1 large forceps, 1 small forceps, 1 large scissors, 1 small scissors.

3. Large (18 mL) plastic Beckman Poly Q scintillation vials (used for digestion of fat to release adipocytes and preadipocytes)

4. Pipettors and tips, 10 mL to 1000 mL Note: for working with primary adipocytes use large bore tips or tips which have been cut to provide a larger opening.

5. Shaking waterbath or incubator with linear shaking platform which can be maintained accurately at 37°C. Example: Adolf Kühner A/G, Lab-Therm 20 LT-W. Note temperature, shaking amplitude and speed are critical for the isolation of adipocytes. A rise in temperature of more than 0.5°C will inevitably result in loss of responsiveness to hormones in the adipocytes.

6. Centrifuge (for hematocrit tubes)

7. Capillary tubes (hematocrit) and sealer

8. pH meter and accessories

9. Pipettes as needed

10. 20 mL syringes with the end with the luer tip cut off to form a tube with plunger

11. Gauze for filtration of cell suspension. 6 x 6 cm squares are cut and autoclaved in 2 layer and three layer packages

12. 50 mL conical polypropylene centrifuge tubes

13. Dialysis tubing

14. 14 ga x 80 mm needles. These needles should reach the bottom of the centrifuge tubes used to recover the adipocytes as they are used to recover the infranatant.

15. Appropriate cell culture growth vessels. ( for growth of preadipocytes)

16. Cell strainer, 70 mm pore size Nylon (Falcon #2350) (for isolation of preadipocytes)

17. Cellector, 25 mm pore size, stainless steel (Bellco Glass, Co. USA) ( for isolation of preadipocytes)

B. Solutions and Reagents

Note: When cells will be cultured after isolation the solutions used should be aseptically filtered. Antibiotics may be added for further protection from contamination in culture.

Note: All solutions/aliquots should be dated and initialed as well as clearly stating the contents.

Note: Water used for all reagents should be reagent grade distilled and/or deionized such as that provided by a Millipore MilliQ system.

Note: All reagents should be aliquoted to avoid repeated freeze/thaw.

1. 139 mM Glucose Stock. (D (+)-glucose)MW = 180.16

Dissolve 2.5 g glucose in 100 mL deionized (H₂O. Aliquot for use and store at

-20°C. Final concentration is 2 mM in wash buffer.

2. 7% BSA Stock Solution (Sigma #A4503)

Dissolve 70 g BSA in 400 mL H₂O. Bring to final volume of 500 mL to give a concentration of 14%.. Dialyse overnight at 4°C against 5 liters of cold H2O with 2 changes of water. After removing from the dialysis bag, adjust pH to 7.4 and bring to a final volume of 1000 mL. Aliquot into 50 mL tubes and store at

-20°C Dialysis removes small molecular weight molecules which might interfere with the responsiveness of the cells and to reduce the influence of contaminating hormones.

3. 100 µM Adenosine Stock. MW 267.2 (optional)

Dissolve 13.4 mg adenosine in 500 mL H₂O. Aliquot into 1.5 mL tubes and store at -20 °C. Final concentration, if used, should be 0.2mM

4. 156.2 mM HEPES Stock, ( Sigma #H3375) MW 238.3

Dissolve 37.234 g Hepes in 900 mL H₂O. Adjust the pH to 7.3 with NaOH. Bring to final volume of 1000 mL. Aliquot and store at -20 °C. Final concentration is 25 mM in collagenase and wash buffers.

5. Collagenase: (Sigma Type II Collagenase, Cat. # C6885)

Collagenase must be tested before purchase by obtaining samples from various suppliers. Collagenase should be handled carefully as it is an irritant and can cause sensitization if inhaled or if it comes in contact with the skin.

Collagenase digestion solution.

May be prepared in advance and stored for up to 1 week.

Final concentration of collagenase is 1 mg/mL in KRH buffer with 3.5% BSA

10 mL 25 mL 50 mL

KR stock (6) 2 mL 5 mL 10 mL

Hepes stock (4) 1.6 mL 4 mL 8 mL

7% BSA stock (2) 5 mL 12.5 mL 25 mL

Glucose stock (1) 144 mL 360 mL 720 mL

Adenosine stock (3) 20 mL 50 mL 100 mL

Collagenase 10 mg 25 mg 50 mg

Mix the stock solutions as indicated and dissolve the collagenase in the mixture. Adjust the pH to 7.4 with NaOH. Bring to the final volume. Place 1.5 mL of the collagenase digestion solution in Beckman vials. Mark with “Collagenase” and date vials. Store at -20 °C. Thaw vials and bring to 37 °C immediately prior to use.

6. KR 5X Stock solution (Krebs Ringers Buffer for use with Hepes, 5X concentrated buffer)

The following makes 1 liter of stock solution.

NaCl 34.70 g

KCl 1.77 g

KH₂PO₄ 0.81 g

MgSO₄ 7H₂O 1.46 g

CaCl₂ ^.4H₂O 2.33 g (alternately use CaCl₂ 2H₂O, 1.87 g)

Dissolve all components except the CaCl₂ in 900 mL H₂O. Once all other salts are dissolved the CaCl₂ may be added. Bring volume to 1 liter. Aliquot and store at

-20°C.

7. Adipocyte Wash Buffer

50 mL 100 mL 200 mL

KR stock (6) 10 mL 20 mL 40 mL

Hepes stock (4) 8 mL 16 mL 32 mL

7% BSA stock (2) 7.1 mL 14.3 mL 28.6 mL

Glucose stock (1) 720 mL 1.44 mL 2.88 mL

Adenosine stock (3) 100 mL 200 mL 400 mL

Adjust pH to 7.4, bring to final volume with deionized water. Note: for storing adipocytes for short periods of time the pH should be adjusted to 7.5.

8. Growth medium, such as DMEM supplemented with antibiotics and fetal bovine serum for culture of preadipocytes

9. Erythrocyte Lysis Buffer ( for culture of preadipocytes)

154 mM NH₄Cl (4.1 g/500 mL)

10 mM KHCO₃ (0.5 g/500 mL)

0.1 mM EDTA (0.019g/500 mL)

C. Protocol for Isolation of adipocytes and preadipocytes:

Note: number of animals used will depend upon the number of adipocytes/preadipocytes needed for the experiments of the day. For isolation of primary adipocytes it is usual to limit the number of animals dissected and processed at any one time to 15-20. For dissection steps 1-3 should be done for each animal as quickly as possible.

Isolation of cells from adipose tissue

1. Sacrifice rats by cervical dislocation

2. Immediately dissect out epididymal fat tissue and place in a scintillation vial with 1.5 mL Collagenase solution for each rat. It is important that the dissection be done carefully with no contaminating tissue such as epididymal gland or blood vessels. If necessary tissue should be rinsed with saline before placing in collagenase solution.

3. Mince tissue in collagenase solution with scissors. Tissue fragments should be 1mm or less and as homogeneous as possible. Usually about 50 strokes of the scissors will be sufficient.

4. Incubate in a shaking waterbath or incubator with linear shaking platform at 37°C for 30-40 minutes. Rate of mixing should be 180 strokes per minute (as judged in Incubator listed above). Exact time is determined empirically and may change with lots of collagenatse. Temperature should not vary more than +/- 1 degree as adipocytes are very sensitive to temperature fluctuations. After the first 30 minutes check digestion visually. Look for free lipids, tissue particles remaining. For the isolation of adipocytes in most cases it is better to have undigested tissue than to allow digestion to go too far. For isolation of preadipocytes only, digestion time can be increased.

5. Place 5 mL of adipocyte wash buffer in a 50 mL conical, polypropylene centrifuge tube.

6. Prepare the filtration unit by putting two layers of gauze over the cut end of the 20 mL tube and securing with a rubber band. Place the gauze end into the 50 mL tube containing the buffer (step 5). Pour the suspension from the vials through the gauze.

7. Rinse the digestion vials by pouring 3 mL wash buffer from vial to vial and then through gauze filter system. Syringe plunger may be used to force fluid through the gauze, however, do not allow the plunger to touch the tissue.

8. Wash gauze with additional 10 mL of wash buffer. Note: syringe plunger may be used to slowly force the material through the gauze, however, do not allow the plunger to touch the tissue fragments. The gauze may be touched against the side of the tube to collect drops of cell suspension.

9. Pour cell filtrate through a second gauze/syringe filter with three layers of gauze and wash with 10 mL wash buffer. Do not use plunger.

10. Discard gauze and remaining tissue fragments.

11. Allow suspension to sit until adipocytes have floated to the top. The fat cells will forma white layer on the top of the solution. Usually 2-3 minutes is adequate. Alternatively, the cell suspension may be centrifuged at 150Xg briefly (start centrifuge, bring to speed then stop) to bring adipocytes to the top. At this point preadipocytes will be in the infranatant.

12. Draw off the collagenase solution (infranatant) using a 20 mL syringe with attached 14 ga. spinal (80mm long) needle. Needle can be put into the tube while the cells are separating or before centrifugation. In this way adipocytes will not be caught on the needle as it is put down into the solution. This infranatant contains preadipocytes as wellas some contaminants (cells and debris). For isolation of pre-adipocytes it should be collected and held until all washes are also collected or preferably centrifuged at 100 g for 5 minutes and the pellet resuspended in medium or wash buffer.

13. Wash adipocytes with an additional 40 mL of wash buffer and repeat steps 11 and 13 three to 4 times, until the infranatant is clear. Note if the primary objective is to obtain preadipocytes and additional wash can be included. This will free additional pre-adipocytes which are bound to the adipocytes. Note: One primary purpose of this wash is to remove the collagenase and therefore, the color of the infranatant can be used to help determine when washing is complete. The initial collagenase solution is faintly brown. Other contaminants will also be separated from the adipocytes with these washes. All washes should be collected and centrifuged at 100 g to pellet preadipocytes as mentioned in step 12.

14. After final wash resuspend adipocytes in appropriate buffer for the next step in the procedure and mix well but gently.

15. Determine the percent vol/vol of the cells in solution using a hematocrit tube. This is done by:

15.1.Place one end of the hematocrit tube in the well mixed cell suspension and allow it to fill about 2/3 of the way.

15.2.Cap the end with clay.

15.3.Centrifuge in a table top centrifuge equipped to spin hematocrit tubes

15.4.Using a ruler determine the total length of the liquid and the portion of the cells. Divide length of cells by total length of liquid and multiply by 100. Calipers may also be used.

Note: Some experiments may require a preincubation of adipocytes at this time or the cells might have to be held for a few minutes. In this case they should be diluted to 5% in wash buffer at pH 7.5, put into a flask and kept at 37 °C with gently shaking ( 60-65 strokes per min). Flask should be such that there is a large surface area and the speed should be such that there is no foaming or splashing, but such that the adipocytes do not collect at the top. Adipocytes are very fragile.

At this point adipocytes are ready for further purification or experimentation, such as cell culture, lipogenesis or other metabolic assays. Additional protocols are available for such assays.

For purification of preadipocytes continue with the protocol below.

Preadipocyte Isolation

1. Continue from step 13 above

2. Centrifuge infranatants if not all ready done.

3. Resuspend the pellets in 13 mL of medium. The pellet can be loosened by running the tube against the bottom of a metal test tube rack before resuspension.

4. Filter cell suspension through a 70 mm pre-wet filter. (Use 5-6 mL medium to pre-wet the filter.

5. Wash filter with 2-3 mL of medium.

6. Filter cell suspension through a 25 mm filter. To avoid loss of cells, pre-wet and wash the filters with medium as above.

7. Centrifuge the cell suspension at 150xG for 10 minutes.

8. Remove supernatant and resuspend the pellet in 25 mL erythrocyte lysis buffer.

9. Let rotate or rock for 10 minutes on rotator or rocker platform

10. Centrifuge the cell suspension at 95 xG for 10 minutes.

11. Aspirate the supernatant and resuspend the pellet in 10 mL fresh growth medium.

12. Count the cells using a hemacytometer and trypan blue.

13. Cells can now be plated at the desired concentration. For differentiation studies 300,000 cells per well in a 6 well dish is an adequate number, however cell number will depend on studies to be undertaken and therefore should be determined empirically.