Cell suspension preparation for flow cytometry and flow cytometry analysis

Isolation of adipose tissue macrophges for analysis by flow cytometry

Materials

1. Reagents
   1. PBS 1X Without Ca²⁺ Mg²⁺
   2. PBS 1X BSA 0.5%, filtered with 0.22μm stericup
   3. CaCl₂ 1mol/L
   4. Facs buffer: PBS 1X + 0.5% BSA + 2mmol/L EDTA, filtered with 0.22μm stericup
   5. Collagenase II (sigma, ref: C6885-1g), prepared at  4 mg/mL in PBS + 0.5% BSA + CaCl₂ 5mmol/L. (For 50mL: 0.2g of collagenase II + 250μL CaCl₂ 1mol/L + 49.75mL PBS 1X + 0.5% BSA)
2. Materials
   1. 6-well plate (Falcon, ref: 353046)
   2. 96-well plate (Thermo scientific, ref: 163320)
   3. 50mL Falcon tubes
   4. 100 μm strainers (Falcon, ref 352360)
   5. 70 μm strainers (Falcon, ref 352350)
   6. 10mL pipettes
   7. 37°C bug incubator, or any other incubator with shaking option
   8. Antibodies – on page 2 -

Procedure

Once the mouse is dead, collect the desired fat pads (for our experiments: mWAT, eWAT, iWAT, iBAT) see reference: Orr et al below – page 3 -

1. Adipose tissues are collected in 2mL of cold PBS, in a 6-well plate on ice. NO more than 1.2g of tissue per well

2. Mince the fat tissues into small pieces and transfer into a clean 50mL falcon tube

3. Wash the wells of the 6-well plate with 1mL PBS and then add to the 2mL already in the 50mL falcon.

4. Add 3mL of Collagenase II mix per tube (final concentration for the collagenase 2 mg/mL)

5. Incubate for 20min on shaker under agitation at 37°C

6. After incubation, add 10mL of ice cold PBS to the falcon tube and pipette up and down several times with a 10mL pipette

7. Filter the cell suspension through a 100μm strainer and then transfer the sample into a new 50 ml falcon tube

8. Spin the sample for 10min at 500g in a swinging-bucket centrifuge

9. Remove supernatant and resuspend the pellet in 50 μL of FACS buffer + FcBlock 

10. Transfer the cell suspension into a 96-well plate and add 50μL of antibody mix, incubate for 30min on ice.

11. Following the incubation period spin down the samples for 7min at 320g in a swinging-bucket centrifuge

12. Wash the cell pellet  with 200μL of FACS buffer 

13. Repeat 3.9 and 3.10 steps 

14. You are now done!! Remember to run the samples through a 70μm strainer prior to flow cytometry analysis

Antibody mix proposition

References

1. Protocol adapted from Orr, Jeb S., Arion J. Kennedy, et Alyssa H. Hasty. 2013. « Isolation of Adipose Tissue Immune Cells », JOVE n^o 75 (mai): e50707. doi:10.3791/50707.
2. For our experiments: mWAT, eWAT, iWAT, iBAT

Schema from Waldén, Tomas B., Ida R. Hansen, James A. Timmons, Barbara Cannon, et Jan Nedergaard. 2012. « Recruited vs. Nonrecruited Molecular Signatures of Brown, “brite,” and White Adipose Tissues ». American Journal of Physiology - Endocrinology and Metabolism 302 (1): E19‑E31. doi:10.1152/ajpendo.00249.2011