Nov 10, 2023
Preparation of Single Cell Suspension from Human Lymph Node Tissue
- Steven B. Wells1,
- Peter A. Szabo2,
- Nora Lam2,3,
- Maya M.L. Poon2,4
- 1Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 2Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 3Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032;
- 4Medical Scientist Training Program, Columbia University Irving Medical Center, New York, NY 10032, USA
- Columbia
Protocol Citation: Steven B. Wells, Peter A. Szabo, Nora Lam, Maya M.L. Poon 2023. Preparation of Single Cell Suspension from Human Lymph Node Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.bwsapeae
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 21, 2021
Last Modified: November 10, 2023
Protocol Integer ID: 51746
Keywords: Lymph node, CD45, Lymphocytes, Myeloid, Isolation, Density gradient, Ficoll, Immune, 10x, scRNAseq, Flow cytometry, Leukocyte, Single cell suspension, T cell, preparation of single cell suspension, single cell suspension, cell suspensions across sample, human lymph node tissue this protocol, cell suspension, human lymph node tissue, progenitors from human lymph node tissue, cell, density centrifugation, dilution factor for density centrifugation, media formulation, defined dilution factor, tissue, method for the isolation
Abstract
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.
Attachments
dzhpbk587.pdf
182KB
Materials
Materials:
- Fisherbrand™ Sterile Syringes for Single UseFisher ScientificCatalog #14955459
- Dulbeccos phosphate-buffered saline (DPBS)Gibco - Thermo Fisher ScientificCatalog #14190144
- Penicillin-Streptomycin-Glutamine (100X)Thermo FisherCatalog #10378016
- Thermo Scientific™ Nunc™ 50mL Conical Sterile Polypropylene Centrifuge TubesFisher ScientificCatalog #12-565-271
- Gibco™ IMDM (Iscoves Modified Dulbeccos Medium)Fisher ScientificCatalog #12-440-053
- Gibco™ Fetal Bovine Serum qualified AustraliaFisher ScientificCatalog #10-099-141
- UltraPure™ 0.5 M EDTA pH 8.0Thermo Fisher ScientificCatalog #15575020
- Thomas ScientificSupplier Diversity Partner Cell Strainer 100um Yellow Sterile Individually WrapFisher ScientificCatalog #50-146-1428
- Ficoll-Paque™ PLUS MediaFisher ScientificCatalog #45-001-749
- Collagenase DMerck MilliporeSigma (Sigma-Aldrich)Catalog #11088882001
- DNASE 1 100MGFisher ScientificCatalog #NC9709009
- Mr. Frosty™ Freezing ContainerFisher ScientificCatalog #5100-0001
- CryoStor CS10 100MLFisher ScientificCatalog #NC9930384
- Corning™ Externally Threaded Cryogenic VialsFisher ScientificCatalog #09-761-71
- 5mL Falcon™ Round-Bottom Polypropylene Test TubesFisher ScientificCatalog #14-959-11A
- Solution 13 AO – DAPIChemometecCatalog #910-3013
- NC-Slide A8™ box with 25 SlidesChemometecCatalog #942-0003
- Falcon™ Plastic Disposable Transfer PipetsFisher ScientificCatalog #1368050
Equipment:
- Multi-Axle-Rotating Mixer/Shaker with Temperature Control
- Centrifuge
- Cell Counter - NC-3000
- Surgical scissors
- Scale
Troubleshooting
Preparing Mediums and Buffers
Create the following IMDM-FBS-PSQ Media in a 500 mL bottle of IMDM by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| IMDM | 500 | - | - | |
| Penicillin-Streptomycin-Glutamine | 5 | 100X | 1X | |
| FBS | 50 | 100% | 10% |
Table 1.
*Final Concentration is approximate.
Create the following DPBS-FBS-EDTA Solution in a bottle of DPBS without calcium and magnesium by using the table below:
| A | B | C | D | |
| Component | Volume (mL) | Starting Conc. | Final Conc.* | |
| DPBS | 500 | - | - | |
| FBS | 25 | 100% | 5% | |
| EDTA | 1 | 0.5M | 1mM |
Table 2.
*Final Concentration is approximate.
Tissue Dissociation
32m
Clean lymph nodes of fat and connective tissue post dissection, record the site below.
___________
Add up to 2 ± 10% grams of cleaned lymph node tissue to per 50 mL centrifuge tube – record the total weight below.
__________g
Note
NOTE: Going beyond the 2 grams of tissue per tube reduces the efficacy of the enzymatic digest and lowers yields.
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”.
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 00:30:00 at 37 °C .
30m
After digestion, add 0.500 mL of EDTA 0.5 Molarity (M) 8.0 to the digested cell suspensions and incubate for 00:02:00 at 20 °C .
2m
Distribute and filter the mash of tissue over 100 micromolar (µM) cell strainers above 50 mL tubes (about 4 filters/2 grams of tissue).
Note
NOTE: Cell yields and ease of pushing through the filter are increased by using multiple filters/gram of tissue, default to using more filters to decrease processing time, and increase yields.
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.
Ficoll-Paque
50m
Centrifuge the cell suspensions for 00:10:00 at 400 x g at 20 °C .
10m
Remove the supernatants and combine the cell pellets down to a single 50 mL tube, top to 50 mL with Room temperature IMDM-FBS-PSQ Media, spike in 0.500 mL of EDTA 0.5 Molarity (M) 8.0 .
Filter the cell suspension through a 100 micromolar (µM) cell strainer.
In two 50 mL tubes, layer 25 mL of cell suspension on top of 15 mL of Ficoll-Paque Media PLUS.
Spin for 00:20:00 , 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).
20m
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 00:10:00 at 400 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in 50 mL cold DPBS-FBS-EDTA Solution and centrifuge the cell suspension for 00:10:00 at 120 x g , 4 °C .
10m
Remove the supernatant and re-suspend the cell pellet in cold 10 mL IMDM-FBS-PSQ Media.
Cell Count
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: ________
Analysis and Freeze-down
(Optional QC) Aliquot 2 x 106 cells to a 5 mL Falcon tube and place on ice for subsequent flow cytometric analysis.
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: __________.