Flow cytometry

Flow cytometry of stromal cells and hematopoietic cells from lymph nodes

Reagents

• PBS

• RPMI-1640 medium (Sigma-Aldrich, cat. no. R8758)

• Fetal Calf Serum (Biowest, cat. no. S1810-050)

• Penicillin-streptomycin (Lonza, cat. no. DE17-602E)

• HEPES buffer solution (PAN Biotech, cat. no. P05-01500)

• EDTA-2Na (Sigma-Aldrich, cat. no. E5134)

• Collagenase P (Roche, cat. no. 12138730001)

• DNase I (Sigma-Aldrich, cat. no. 4527)

• Dispase (Roche)

Equipment

• Flow cytometer

• Constant-temperature incubator/shaker

• 15-ml tube (BD Falcon, cat. no. 352096)

• 50-ml tube (BD Falcon, cat. no. 352070)

• 1.5 ml tube (Biomedical sciences, cat. no. BC-MPF-150C)

• 5-ml polystyrene round-bottomed FACS tubes (BD Biosciences, cat. no. 352058)

• 1 ml cut tips

• 24 well culture plates (TPP, cat no. 009224)

• 96 well round bottom (Greiner Bio-one, cat. no. 7.650101)

• 3 ml syringes (BD Plastipak)

• 26 G needles (Terumo)

• 70 μm nylon cell strainer (BD Falcon, cat. no. 340336)

Prepare:

1. Digestion medium

   RPMI 2% of FCS

   20mM HEPES

2. Digestion medium + enzymes

   Dispase (40 µg/ml)

   Collagenase P (1 mg/ml)

   DNAse (25 µg/ml)

   24 well plate with 1ml of Digestion medium without enzymes/well. One well is needed per lymph node.

    

Preparation of lymph node stromal cells

1. Sacrifice the mice and harvest the lymph nodes. Transfer them in to the 24 well plate containing digestion medium without enzymes.

2. Replace the medium without enzymes with 1 ml of with pre-warmed digestion medium + enzymes (37°C).

3. Disrupt the lymph nodes using 26G needles to make small pieces.

4. Transfer the disrupted organs in to a fresh 15ml Falcon tube using cut tips.

5. Incubate 15 min in 37°C water bath; do not incubate cells for longer.

6. During the incubation, prepare another set of fresh 15mL Falcon tubes with 5ml of MACS buffer (2% FCS, 3mL of EDTA in PBS 1X). Place the tubes on ice.

7. After incubation, disrupt gently the tissue by pipetting up and down using 1 ml cut tips (approx. seven times). Wait 2-3 minutes to let the nondigested tissue go to the bottom of the tube. Transfer the supernatant to the tube containing MACS buffer. Refill the tube  with 1 ml of pre-warmed digestion medium + enzymes.

8. Incubate 10 min in the water bath and repeat step 7 until no pieces of tissue are visible. Normally it takes three rounds of digestion to get a single cell suspension. After the second round of digestion, normal 1 ml tips can be used for tissue dissociation.

9. Centrifuge the tubes 1200 RPM 5 min.

10. Discard the supernatant and resuspend the pellet in 1ml of MACS buffer.

11. Add 50 µL of anti-CD45 and anti-Ter119 MACS beads.

12. Incubate 20 min on ice.

13. Wash with 5 ml of MACS buffer and centrifuge at 1200 RPM 5 min.

14. In the meantime, activate the MACS columns with 3 ml of MACS buffer. Add collection tubes in the columns. Important the negative population contains the stromal cells.

15. Resuspend the cells in 5 ml of MACS buffer and add the cells to the top of the MACS column.

16. Wash 2 times with 3 ml of MACS buffer. Collect the negative population (Stroma cells)

17. Centrifuge the negatively selected cells (CD45 ter119 depleted cells) 1200 RPM 5 min. Discard the supernatant.

18. Stain the cells with the viability dye 1:1000 in PBS for 20 min at 4 °C. Wash the cells by adding 5 ml of MACS buffer. Centrifuge the samples at 1200 RPM for 5 min. discard the supernatant.

19. Incubate the cells for 20 minutes at 4 °C with the desired labelled antibodies diluted in MACS buffer. See Table S1

20. Wash the cells by adding 5 ml of MACS buffer and centrifuge the samples 1200 RPM 5 min.

21. Cells are ready for flow cytometric analysis.

Preparation of hematopoietic cells

1. Sacrifice the mice and harvest the lymph nodes. Transfer them in to the 15 ml Falcon tubes containing 10 ml of PBS.

2. Smash the organs through a 70 µm filters using a 3 ml syringe plunger.

3. Transfer the cells to a 15 ml Falcon tube.

4. Centrifuge the 10 ml cell suspension at 1500 RPM 5 min.

5. Cell are ready for staining. (Check Table S1 to adapt your staining of interest).

Flow cytometry analysis shown in the manuscript was performed a FACS Canto or a BD LSRFortessa (BD Biosciences) and analyzed using FlowJo software (Treestar Inc.)

Table S1. Antibodies used in the study.

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