TITLE: Preparation of Single Cell Suspension from Human Spleen Tissue
AUTHORS: Steven B. Wells, Peter A. Szabo

[DESCRIPTION]
This protocol describes a method for the isolation of pan-lymphocytes, pan-myeloid cells, and progenitors from human spleen 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 Medium and Buffer
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 Medium and Buffer
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. Add 2 ± 10% grams of spleen tissue to a 50 mL centrifuge tube and record below.
__________g
SECTION: Tissue Dissociation
4. Add 5 mL of DPBS-FBS-EDTA Solution to the spleen tissue and use a scissors to chop the tissue into a fine “mash”.
  

SECTION: Tissue Dissociation
5. Add 35 mL of DPBS-FBS-EDTA Solution to the mash of tissue, and distrubute and filter the tissue over 100 micromolar (µM) cell strainers above 50 mL tubes (about 4 filters/2 grams of tissue).
 

SECTION: Tissue Dissociation
6. 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 pink/white connective tissue remains. When finished, combine the tubes of cell suspension and proceed to the next section.

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

SECTION: Ficoll-Paque
8. Remove the supernatants and combine the cell pellets down to a single 50 mL tube, top to 50 mL with 20 Room temperature DPBS-FBS-EDTA Solution.

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

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

SECTION: Ficoll-Paque
11. 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
12. 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
13. Remove the supernatants and re-suspend the cell pellets 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
14. Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.

SECTION: Cell Count
15. 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: Freeze-down and QC
16. (Optional QC) Aliquot 2 x 106 cells to a 5mL Falcon tube and place on ice for subsequent flow cytometric analysis.
SECTION: Freeze-down and QC
17. 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 30 million cells and discard/freeze/use any left over cells). Record the number of vials frozen: __________.