Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.
B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.
Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the naïve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells.
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors.
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify naïve B cells or T cells.
All mice are bred and maintained under specific pathogen-free conditions and all mouse protocols are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.
1. Preparation of Buffers and Reagents
2. Generation of Lymphocyte Suspension from Spleen or Lymph Nodes
NOTE: It is important to prepare all reagents and equipment required for the experiment before mouse euthanasia and to generate single cell suspensions of lymphocytes as soon as possible to maintain high cell viabilities.
3. Purification of B and T Cells
4. CFSE Labeling and Stimulation
NOTE: Purified cells can be subjected to a variety of in vitro and in vivo functional assays. Here, we use purified T cells to determine the T cell stimulation capability of APCs5.
5. In Vitro Stimulation
For B cells | |
Stimuli | Final concentration |
F(ab’)2 goat anti-mouse IgM | 0.6-2.4 µg/ml |
Anti-mouse CD40 mAb | 0.5-2 µg/ml |
Recombinant mouse IL-4 | 25 U/ml |
Lipopolysaccharide | 0.1-10 µg/ml |
(LPS) from E. coli Serotype 055:B5 | |
For T cells | |
Stimuli | Final concentration |
Anti-CD3 (plate coated) | 2-10 µg /ml |
(50 µl/well for coating) | |
Anti-CD28 (plate coated) | 2 µg/ml |
Recombinant IL-2 | 40 U/ml |
PDBu (Phorbol ester) | 5-50 ng/ml |
A23187 (Calcium ionophore) | 250 ng/ml |
Table 1: Concentrations of stimuli used to stimulate lymphocytes in in vitro culture.
6. In Vivo Stimulation
Magnetic cell purification of lymphocytes allows users to purify a target cell population in a relatively short amount of time. Using our depletion protocol, we were able to increase the percentage of CD8 T cells (OT-I in recombination-activating gene-1 (RAG-1)-deficient mice) from 72.8% (before purification) to 94.2% (after purification; Figure 1A)4,5. These purified lymphocytes can then be used for downstream functional assays to determine lymphocyte proliferation and signal transduction4,5. For example, we can study the in vivo T cell stimulation capacity of APCs by transferring CFSE-labeled, antigen-specific T cells into wildtype (WT) and mutant (MT) mice immunized with suitable antigen5.
In our representative experiment, we transferred purified, CFSE-labeled, ovalbumin (OVA) specific OT-I CD8 T cells into control (WT) and mutant (MT) mice and immunized these mice one day later with OVA protein. Three days after immunization, we harvested lymph nodes (proximal and distal) and spleens and analyzed the T cells by flow cytometry. CD45 allelic forms can be utilized to better separate the dividing, CFSE-labeled, donor OT-I CD8 cells from non-labeled, recipient CD8 T cells. In this example, donor and recipient cells are from CD45.1 and CD45.2 mice, respectively (Figure 1B). The CFSE levels of surviving, unstimulated OT-I T cells at this time point are used to define the peak for non-proliferating cells (Figure 1C, dotted line). Upon stimulation, the intensity of the CFSE levels in OT-I T cells will reduce by half with each division. T cell proliferation can thereby be determined by counting the number of CFSE peaks6,12. In our representative results, we do not see differences in the cross-presentation capacities of WT and MT APCs (Figure 1C, solid lines), since the T cells proliferate at similar rates in both mice.
In a separate experiment, we performed a [3H]-thymidine incorporation assay to study the cell proliferation of activated control and MT T cells. Purified WT and MT T cells were stimulated with various stimuli for 48 hr and pulsed with [3H]-thymidine for the final 8 hr. Proliferating cells in the S-phase of the cell cycle will incorporate the radiolabeled nucleotide, [3H]-thymidine, into newly synthesized deoxyribonucleic acid (DNA), therefore, using liquid scintillation counting, cell proliferation can be measured by [3H]-thymidine uptake. The number of counts per minute (c.p.m.) directly correlates with the amount of [3H]-thymidine uptake in proliferating T cells. In our representative results, the reduced number of c.p.m. obtained from MT T cells upon stimulation compared to counts from WT T cells indicates a compromised proliferative capacity of MT T cells (Figure 1D).
To determine B cell proliferative capacity, we purified splenic B cells using the described depletion strategy. Upon purification, we were able to increase the percentage of B220 B cells (WT mice) from 63.9% (before purification) to 98.4% (after purification; Figure 2A)4. Similar to purified T cells, purified splenic B cells can also be used for downstream functional assays to assess lymphocyte proliferation and signal transduction4. Subsequently, we labeled purified WT splenic B cells with CFSE and stimulated these cells in vitro using plates coated with anti-CD40 supplemented with the cytokine IL-4. Three days after stimulation, we analyzed viable, activated B cells by flow cytometry. The CFSE levels of surviving, unstimulated B cells at this time point are used to define the peak for non-proliferating cells (Figure 2B, dotted line). Similar to T cells, the intensity of the CFSE levels in B cells will reduce by half with each division6,12.
Figure 1: CFSE profiles of adoptively transferred purified OT-I T cells after immunization. (A) CD4 and CD8 staining of cells isolated from OT-I; RAG-1-deficient mice before (upper panel) and after (lower panel) non-T cell depletion. (B) Representative FACS plots of T cells from spleen (Sp), proximal lymph nodes (pLN) and distal lymph nodes (dLN). Donor OT-I CD8 T cells are CD45.1 positive. (C) Representative CFSE profiles of transferred, CFSE-labeled OT-I donor T cells from spleen (Sp), proximal lymph nodes (pLN) and distal lymph nodes (dLN) of WT (shaded curves) and MT (solid lines) recipient mice 3 days after immunization with OVA protein and LPS. Dotted line indicates OT-I T cells from WT recipient mice without immunization (PBS injected). (D) Cell proliferation of activated control or MT T cells as measured by [3H]-thymidine uptake assay. Results are presented in counts per minute (c.p.m.). Purified control (open bar) or MT (closed bar) T cells were incubated for 48 hr in medium only (medium) or in the presence of plate-bound anti-CD3 or anti-CD3 plus anti-CD28 with or without recombinant IL-2. Polyclonal cell activation was triggered by PMA (phorbol ester) and A23 (calcium ionophore). Please click here to view a larger version of this figure.
Figure 2: CFSE profile of B cells upon in vitro stimulation. (A) B220 and CD3 staining of splenic cells isolated from WT mice before (upper panel) and after (lower panel) non-B cell depletion. (B) Representative CFSE profile of CFSE-labeled WT (shaded curve) splenic B cells after 3 days of in vitro stimulation with plates coated with anti-CD40 and IL4. Dotted line indicates CFSE-labeled WT B cells without stimulation. Please click here to view a larger version of this figure.
In this protocol, we demonstrate a procedure for purifying lymphocytes from lymphoid organs. Cell purification using magnetic bead sorting is a fast and simple method that yields viable, highly purified target cells.
Critical Steps within the Protocol
Cell viability and cell yield
Maintaining viability of hematopoietic lineage cells in vitro is critical to ensuring successful and reproducible experiments. Chemical and biological reagents, sub-optimal experimental conditions or improper storage conditions of excised organs can all affect cell viability. Upon excision from mice, lymphoid organs need to be stored on ice and single cell suspensions prepared as soon as possible. High-speed centrifugation of cell suspensions should also be avoided. Furthermore, cell pellets should be loosened by flicking tubes with fingers after removal of supernatant. It is not recommended to re-suspend pellets directly with large amounts of medium by pipetting.
Approximately 2-4 x 107 splenic B cells and 2 x 107 T cells from all major lymph nodes can be isolated from one 8-10 week-old WT mouse. Reduced numbers of purified B and T cells (below the expected value) indicates sub-optimal conditions, as mentioned earlier.
Cell purity
The average purity of isolated cells using this protocol is within the range of 90 to 95%, which is sufficiently pure for subsequent in vitro or in vivo experiments (Figure 1A). It is advisable not to overload the separation column with excessive numbers of cells during the process of separation because doing so can compromise cell purity due to the column's limited binding capacity.
Quality of FBS
The quality of FBS used to supplement the culture medium is critical for the in vitro survival of lymphocytes. FBS from different sources and batches may vary in their ability to support in vitro lymphocyte responses. Therefore, it is important to test different types of FBS to find one that gives high-specific response with low background. A substantial number of bottles should then be reserved as a stock.
Modifications and Troubleshooting
CFSE labeling conditions
Even though CFSE labeling works in plain RPMI, we have found that using a low percentage of FBS in PBS reduces cell death during the loading process, without compromising the efficiency of labeling. Moreover, the presence of FBS minimizes cell loss during centrifugation.
An important aspect of CFSE labeling is to ensure even labeling of the cells in order to visualize the distinct peaks that represent cell division. Overloading of CFSE could result in increased cell death in vitro or poor recovery of labeled cells in vivo. On the other hand, insufficient CFSE labeling or heterogeneity in the target cell suspension during the process of labeling can result in poorly resolved CFSE peaks12. Therefore, it is important to optimize the CFSE labeling conditions. The amount of CFSE used for labeling should be kept as low as possible to reduce potential toxic effects from overloading, but still achieve sufficient labeling to detect nicely resolved peaks upon stimulation within the experimental time frame. In addition, to avoid poorly resolved peaks, cell clumps should be removed from single cell suspensions prior to CFSE loading.
Cell clumps and cell loss
It is crucial to ensure that cells are fully re-suspended before proceeding to the next step because the perpetual removal of cell clumps during the experiment drastically reduces cell numbers. Cell clumps are usually associated with reduced cell viability or inadequate re-suspension of the cell pellet.
CFSE and emission spectra overlap
CFSE-labeled cells can be further defined with fluorophore-conjugated antibodies after one day in culture, however, these CFSE-labeled cells still remain brightly fluorescent after a day in culture. Using combinations of antibodies conjugated to bright fluorophores with minimal amounts of emission spectra overlap with CFSE, such as phycoerythrin (PE) and phycoerythrin-cyanine 7 (PeCy7), provides optimal staining results. However, compensation to reduce emission spectra overlap is still required. Additionally, due to the high intensity of CFSE signal (FL-1 channel) that spills into the FL-2 channel, the FL-2 channel has to be compensated by deducting the high percentage of FL-2 value to achieve an optimal CFSE profile. One can refer to the paper published by Quah et al., which provides solutions for troubleshooting CFSE labeling and analysis of results12.
Alternatives to CFSE
The emission spectrum of CFSE restricts the use of combinations of CFSE with fluorescein derivatives such as green fluorescent protein (GFP) and fluorescein isothiocyanate (FITC)-conjugated antibodies. There are, however, other commercially available cell dyes with equally high fluorescence intensity and low cell toxicity in violet (CTV, excitation/emission: 405/450 nm), yellow (CTY, 555/580 nm), far red (CTFR, 630/661 nm or CPD670, 647/670 nm) and red (PKH26, 551/567 nm). Using cell labeling dyes with different emission spectra provides flexibility in experimental design. On the other hand, not all of the above-described cell labeling dyes can generate distinct cell division peaks. A comparative study performed using various cell labeling dyes concluded that CTV is a better replacement for CFSE because CTV enables the detection of a higher number of clearly defined cell division peaks13.
Limitations
The major limitation of magnetic cell sorting is that it is only suitable for simple cell sorting. In our example, we could use CD43 to deplete all non-B cells from spleen; however, we would not be able to separate follicular B cells from marginal zone B cells. These sub-populations can only be defined with multiple surface markers. While it is possible to positively select cells using magnetic cell sorting based on multiple surface markers, it is dependent on specially prepared, commercially available reagents that enable the release of microbeads from target cells after the first round of sorting before proceeding to the second marker. Thus, the user would be completely dependent on the commercially available kits. In such a case, FACS is much more advantageous in separating refined cell populations.
Significance of Technique
Antibody-based cell sorting approaches, such as magnetic cell sorting and FACS, are the most reliable cell sorting techniques to date15. Other methods of cell separation exist, including density-based and adherence-based techniques, however, lymphocytes are poorly adherent cells and their subpopulations are relatively similar in density, thus adherence- and density-based techniques are either not applicable or are very inefficient15.
As mentioned in the introduction, rapid, simple, high cell viability, and independent of any sophisticated equipment are the winning features of magnetic cell sorting over FACS. In particular, negative depletion using magnetic cell sorting labels and depletes undesirable cells using a magnetic separation column, while isolating target cells with minimal modifications to the cell surface and maintaining the naïve state.
Future Applications
The highly enriched, viable, and naïve lymphocytes purified using this magnetic-based purification technique can be subjected to various functional assays of lymphocyte behavior and signaling mechanisms in vitro and in vivo. In this protocol, we demonstrated using two different cell proliferation assays — [3H]-thymidine incorporation assay and CFSE cell proliferation assay — to investigate the cell proliferative capacities of activated lymphocytes.
The choice between [3H]-thymidine incorporation assay and CFSE cell proliferation assay depends largely on sample size and experimental conditions. Utilizing the [3H]-thymidine incorporation assay in experiments with large sample sizes generates high-throughput cell proliferation data. In order to ensure optimal [3H]-thymidine uptake and reproducibility of results, [3H]-thymidine should be added to the culture when a majority of the cells are actively dividing. Optimization of the labeling protocol is required for different cell types and stimuli. Furthermore, due to the radioactive nature of [3H]-thymidine, this incorporation assay can only be performed in vitro, unlike CFSE labeling of cells, which can be activated in vitro (Figure 2B) or adoptively transferred into recipient mice (Figure 1B and 1C).
The CFSE cell proliferation assay, on the other hand, offers more information on cell proliferation than [3H]-thymidine incorporation. Since the intensity of CFSE in CFSE-labeled cells reduces by half with each cell division, one can determine the number of cell divisions and the proportion of cells at each division by counting the number and size of peaks12,13. Furthermore, CFSE-labeled cells can be further defined into different subsets according to their surface molecule expression by flow cytometry using the corresponding fluorophore-conjugated antibodies. Most importantly, unlike the [3H]-thymidine incorporation assay that measures cell proliferation at the final time point, the CFSE cell proliferation assay allows for the tracking of cell division, providing more information on the kinetics of cell proliferation capacity. However, the CFSE cell proliferation assay may not be suitable for experiments with large sample sizes.
The authors have nothing to disclose.
The study is supported by the Ministry of Education, Singapore (AcRF Tier1-RG40/13 and Tier2-MOE2013-T2-2-038). The manuscript was edited by Amy Sullivan from Obrizus Communications.
Materials | |||
RPMI 1640 (without L-Glutamine) | Gibco | 31870025 | |
Fetal Bovine Serum | Heat inactivated | ||
L-glutamine | Gibco | 25030024 | |
Penicillin/Streptomycin | Gibco | 15140114 | |
2-mercaptoethanol | Gibco | 21985023 | |
Anti-CD43 magnetic microbeads | Miltenyi Biotec | 130-049-801 | Mix well prior use |
Streptavidin microbeads | Miltenyi Biotec | 130-048-101 | Mix well prior use |
Anti-Annexin V magnetic beads | Miltenyi Biotec | 130-090-201 | Mix well prior use |
MACS LD | Miltenyi Biotec | 130-042-901 | |
96-well U-bottom sterile culture plate | Greiner Bio-one | 650180 | |
96-well F-bottom sterile culture plate | Greiner Bio-one | 655180 | |
100 μm cell strainer mesh | To sterilize using UV radiation prior use | ||
0.2 μm sterile disposable filter units | Nalgene | 567-0020 | Can be substituted with any sterile filter device |
CellTrace Violet | Invitrogen | C34557 | CTV for short; alternative to CFSE |
CellTrace Yellow | Invitrogen | C34567 | CTY for short; alternative to CFSE |
CellTrace Far Red | Invitrogen | C34564 | CTFR for short; alternative to CFSE |
Cell Proliferation Dye eFluor 670 | eBioscience | 65-0840 | CPD670 for short; alternative to CFSE |
PKH26 | Sigma Aldrich | PKH26GL | PKH26, alternative to CFSE |
Name | Company | Catalog Number | Comments |
Chemicals | |||
Dextrose | Sigma Aldrich | G7021 | |
Potassium phosphate monobasic | Sigma Aldrich | P5655 | |
Sodium phosphate dibasic | Sigma Aldrich | S5136 | |
Phenol Red | Sigma Aldrich | P0290 | |
Calcium chloride dihydrate | Sigma Aldrich | C7902 | |
Potassium chloride | Sigma Aldrich | P5405 | |
Sodium chloride | Merck Millipore | S7653 | Can use from other sources |
Magnesium chloride hexahydrate | Sigma Aldrich | M2393 | |
Magnesium sulfate | Sigma Aldrich | M2643 | |
Ammonium chloride | Sigma Aldrich | A9434 | |
Tris-base | |||
Dimethyl Sulfoxide | Sigma Aldrich | D8418 | |
(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) | Molecular Probes | C-1157 | Reconstitute in DMSO |
Phorbol 12,13-dibutyrate (PBDU, Phorbol ester) | Sigma Aldrich | P1269 | |
A23187 (Calcium ionophore) | Sigma Aldrich | C7522 | |
Name | Company | Catalog Number | Comments |
Antibodies and recombinant protein | |||
CD11b biotin (clone m1/70) | Biolegend | 101204 | T cell depletion cocktail |
CD11c biotin (clone N418) | Biolegend | 117304 | T cell depletion cocktail |
Gr-1 biotin (clone RB6-8C5) | Biolegend | 108404 | T cell depletion cocktail |
Ter119 biotin (clone Ter119) | Biolegend | 116204 | T cell depletion cocktail |
TCR-γδ biotin (clone GL-3) | Biolegend | 118103 | T cell depletion cocktail |
CD19 biotin (clone 6D5) | Biolegend | 115504 | T cell depletion cocktail |
B220 biotin (clone RA3-6B2) | Biolegend | 103204 | T cell depletion cocktail |
CD49b biotin (clone DX5) | Biolegend | 108904 | T cell depletion cocktail |
CD4 biotin (clone GK1.5) | Biolegend | 100404 | T cell depletion cocktail |
CD8 biotin (clone 53-6.7) | Biolegend | 100704 | T cell depletion cocktail |
F(ab’)2 goat anti-mouse IgM (plate coated) | Jackson ImmunoResearch | 115-006-075 | 50 µl/well for coating (96-well) |
Anti-mouse CD40 mAb (plate coated) | Pharmingen | 553722 | 50 µl/well for coating (96-well) |
Recombinant IL-4 | ProSpec | Cyt-282 | |
LPS from E. coli Serotype 055:B5 | Sigma Aldrich | L-4005 | |
Anti-CD3 (clone clone OKT3) (plate coated) | eBioscience | 16-0037-85 | 50 µl/well for coating (96-well) |
Anti-CD28 (clone clone 37.51 ) (plate coated) | eBioscience | 16-0281-85 | 50 µl/well for coating (96-well) |
Recombinant IL-2 | ProSpec | Cyt-370 | |
Albumin from chicken egg white, Ovalbumin | Sigma Aldrich | A7641 |