TITLE: Preparation of Single Cell Suspension from Human Lymph Node Tissue
AUTHORS: Steven B. Wells, Peter A. Szabo, Nora Lam, Maya M.L. Poon

[DESCRIPTION]
This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human lymph node tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

[STEPS]
SECTION: Preparing Mediums and Buffers
1. Create the following IMDM-FBS-PSQ Media in a 500 mL bottle of IMDM by using the table below:
 
      Component  Volume (mL)  Starting Conc.   Final Conc.*    IMDM  500  -  -    Penicillin-Streptomycin-Glutamine  5  100X  1X    FBS  50  100%  10%  
 
SECTION: Preparing Mediums and Buffers
2. Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS without calcium and magnesium by using the table below: 
      Component  Volume (mL)  Starting Conc.   Final Conc.*    DPBS  500  -  -    FBS  25  100%  5%    EDTA  1  0.5M  1mM  
 
SECTION: Tissue Dissociation
3. Clean lymph nodes of fat and connective tissue post dissection, record the site below.
___________
SECTION: Tissue Dissociation
4. Add up to 2 ± 10% grams of cleaned lymph node tissue to per 50 mL centrifuge tube – record the total weight below.
__________g
 

SECTION: Tissue Dissociation
5. Add 5 mL of room temperature IMDM (NO ADDITIVES! Just the base media formulation) to each tube and use a scissors to chop the tissue into a fine “mash”.

SECTION: Tissue Dissociation
6. Add 35 mL of room temperature IMDM (NO ADDITIVES! Just the base media formulation) and spike in 0.400 mL of Collagenase D, and 0.400 mL of DNAse to the tube to begin the enzymatic digestion. Place on a shaker or rotator for 30 min at 37 °C.

SECTION: Tissue Dissociation
7. After digestion, add 0.500 mL of EDTA 0.5 Molarity (M) pH 8.0 to the digested cell suspensions and incubate for 2 min at 20 °C.
  

SECTION: Tissue Dissociation
8. Distribute and filter the mash of tissue over 100 micromolar (µM) cell strainers above 50 mL tubes (about 4 filters/2 grams of tissue).
 



SECTION: Tissue Dissociation
9. Apply pressure with the black rubber bottom or the plastic end of a 10 mL syringe plunger to any remaining, partially digested tissue on the cell strainers, and intermittently wash through with DPBS-FBS-EDTA Solution from a transfer pipet – the aim is to push and wash through the tissue until only light pink/white/grey connective tissue remains. When finished, combine the tubes of cell suspension and proceed to the next section.

SECTION: Ficoll-Paque
10. Centrifuge the cell suspensions for 10 min at 400 x g at 20 °C.

SECTION: Ficoll-Paque
11. Remove the supernatants and combine the cell pellets down to a single 50 mL tube, top to 50 mL with 20 Room temperature IMDM-FBS-PSQ Media, spike in 0.500 mL of EDTA 0.5 Molarity (M) pH 8.0.

SECTION: Ficoll-Paque
12. Filter the cell suspension through a 100 micromolar (µM) cell strainer.

SECTION: Ficoll-Paque
13. In two 50 mL tubes, layer 25 mL of cell suspension on top of 15 mL of Ficoll-Paque Media PLUS.

SECTION: Ficoll-Paque
14. Spin for 20 min, 1200 x g at 20 °C with 4 acceleration and 0 brake, evenly distribute the tubes across the entire rotor to prevent wobbling (use all four buckets if possible as opposed to just two).

SECTION: Ficoll-Paque
15. Remove the mononuclear cell layer from both tubes with a transfer pipet and combine in one 50 mL tube. Add cold DPBS-FBS-EDTA Solution to a final volume of 50 mL and centrifuge the cell suspension for 10 min at 400 x g, 4 °C.

SECTION: Ficoll-Paque
16. Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 10 min at 120 x g, 4 °C.

SECTION: Ficoll-Paque
17. Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.

SECTION: Cell Count
18. Count cells, and viability by using the NC-3000 cell counter. Calculate total viable cells and record below:
cell number: ________cells/mL, ________% viable
final volume: ________mL
𝑐𝑒𝑙𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 (𝑐𝑒𝑙𝑙𝑠/𝑚𝐿) ∗ 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦(%) ∗ 𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒(𝑚𝐿) = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
Total Viable Cells: ________
SECTION: Analysis and Freeze-down
19. (Optional QC) Aliquot 2 x 106 cells to a 5 mL Falcon tube and place on ice for subsequent flow cytometric analysis.

SECTION: Analysis and Freeze-down
20. Aliquot cells for analysis or experimentation, and then freeze down cells in up to 3 x 107 aliquots using Cryostor CS10 Medium, a Mr. Frosty, and a -80 °C freezer (1 mL-1.5 mL aliquots, round down to the nearest 20 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.