Isolation of Murine Natural Killer Cells
UNIT 3.22
Melissa A. Pak-Wittel,1 Sytse J. Piersma,1 Beatrice F. Plougastel,1 Jennifer Poursine-Laurent,1 and Wayne M. Yokoyama1,2 1
Division of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 2 Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry. Curr. Protoc. Immunol.. 105:3.22.1C 2014 by John Wiley & Sons, Inc. 3.22.9. Keywords: immunology r innate immunity r cell isolation r magnetic separation
This unit describes the isolation of natural killer (NK) cells from mouse spleen. NK cells represent the third major population of lymphocytes (Yokoyama, 2013). Although NK cells were initially characterized by their tumor-killing capacity, it is now appreciated that they perform essential functions in early phases of immune response to both viral and bacterial pathogens (Horowitz et al., 2011; Vidal et al., 2011). Following activation (see UNIT 11.9B), NK cells can kill cells infected with certain intracellular pathogens, such as Cytomegalovirus, and produce cytokines that orchestrate the subsequent development of specific immunity (Scalzo et al., 2007). Moreover, the pathogen may modulate NK cell and other immune responses such that analysis of NK cell function frequently requires the study of isolated, highly purified NK cell populations. Consideration should be given to generation of interleukin 2 (IL-2)–activated NK cells [also known as lymphokineactivated killer (LAK) cells], because larger numbers are readily produced by in vitro expansion. IL-2 exposure, however, results in differences owing to activation, such as NK cell killing of a broader panel of targets, which may be inappropriate for certain experimental settings. Because NK cells constitute 2.5% of mouse splenocytes (based on NK1.1+ CD3ε− phenotype in C57BL/6 mice; Hackett et al., 1986), the isolation of purified, freshly separated NK cells poses a particular challenge. In this protocol, a method for preparing a highly purified NK cell population from mouse spleen is described. The goal is to eliminate the contaminating T cell, B cell, dendritic cell, granulocyte, and monocyte populations. This is accomplished by depleting the contaminating cells, which have been labeled with selected biotinylated monoclonal antibodies (MAbs) and magnetic beads. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, will not be functionally affected by antibody cross-linking. Additionally, no one “inert” marker has been identified that is expressed specifically on all subsets of NK cells; therefore, negative selection allows for enrichment of all NK cell subsets instead of a specific subset. Purity can then be assessed by cell surface phenotype. The MAb PK136 is directed against the Current Protocols in Immunology 3.22.1-3.22.9, April 2014 Published online April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142735.im0322s105 C 2014 John Wiley & Sons, Inc. Copyright
BASIC PROTOCOL
In Vitro Assays for Mouse Lymphocyte Function
3.22.1 Supplement 105
cell surface molecule NK1.1 (NKR-P1C), one of the most specific markers on NK cells in C57BL/6 mice. Although a small population of NKT cells express NK1.1, NK cells should be distinguished from T cells because the NK cells are surface CD3ε− . Thus, this protocol results in an unstimulated, highly purified CD3ε− , NK1.1+ NK cell population that can be used to perform functional assays. Note that the protocol is optimized for spleen cells from C57BL/6 or C57BL/10 mice because of the detection of NK1.1+ CD3ε− NK cells in the C57BL background and due to the high prevalence of H2 congenic and genetically modified strains on this background.
Materials C57BL/6- or C57BL/10 mice Tris/NH4 Cl lysing buffer (see recipe) Complete RPMI-10 (R10) medium (see recipe) Fc blocking buffer (see recipe) or a commercially available Fc-blocking agent Isolation buffer (see recipe) 0.5 mg/ml biotinylated MAbs (eBioscience): CD3ε (145-2C11), CD8a (53-6.7), CD4 (GK1.5), CD14 (Sa2-8), CD19 (eBio1D3), TER-119, and Ly6G (GR-1) Anti-biotin microbeads (Miltenyi Biotech) Anti-biotin Dynabeads for alternative separation protocol Fluorophore-conjugated MAbs for flow cytometry: CD3ε-FITC, NK1.1-(PK136)-PerCP-Cy5.5, CD19-PE-Cy7, MHC Class II (I-A/I-E)-AlexaFluor700, CD11c-APC-Cy7, CD11b-eFluor450 (eBioscience), Ly6G (1A8)-PE and F4/80 (BM8)-AlexaFluor647 (Biolegend) Fixable cell viability marker eFluor506 for flow cytometry (eBiosciences) 70-μm cell strainer (BD Biosciences) 50- and 15-ml conical screw-cap polypropylene centrifuge tubes Beckman GS-6R swinging-bucket centrifuge with GH3.8 rotor (or equivalent) Platform rocker LS column and magnetic separator (Miltenyi Biotec; also see UNIT 3.5A) DynaMag-15 (Life Technologies) for alternate separation protocol. 40-μm pre-separation filter (Miltenyi Biotec) Additional reagents and equipment for euthanasia of mice (UNIT 1.8), removal of mouse spleen (UNIT 1.9), preparation of a cell suspension from mouse spleen (UNIT 3.1), red blood cell lysis (UNIT 3.1), use of nylon wool column for removing macrophages and dendritic cells (UNIT 3.2), counting of viable cells by trypan blue exclusion (APPENDIX 3B), antibody titration (UNIT 5.3), magnetic bead separation (UNIT 3.5A), and flow cytometry (Chapter 5) NOTE: All reagents and materials used in the preparation of these cells must be sterile if cells are to be used in culture.
Preparation of splenocyte suspension in 50-ml polypropylene centrifuge tubes 1. Euthanize mice (UNIT 1.8), and aseptically remove spleen (UNIT 1.9). 2. Prepare splenocyte suspension as described in UNIT 3.1, except use a 70-μm cell strainer, transferring cells into 50-ml polypropylene centrifuge tubes. Avoid transferring fibrous debris to the collection tube.
3. Wash cells by centrifugation 5 min at 585 × g, 4°C.
Isolation of Murine Natural Killer Cells
4. Lyse red blood cells as described in UNIT 3.1, except use 1 ml of Tris/NH4 Cl lysing buffer per spleen and, after quenching the reaction with R10 medium, filter cells through a 70-μm cell strainer into a new 50-ml conical tube. 5. Resuspend cells in R10, using 1 ml per spleen.
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6. Load and run nylon wool column as described in UNIT 3.2. Because the nylon wool column removes macrophages and dendritic cells, be careful to not overload the column. Generally, 1.5 × 108 cells for a 2-ml nylon wool column bed is ideal. For larger cell quantities, increase the number of wool columns appropriately. There is a 30% to 50% loss of total cell number on a nylon wool column.
7. Centrifuge flowthrough collected from nylon wool columns 5 min at 585 × g, 4°C. Aspirate supernatants, resuspend pellets, and, if multiple columns were used, combine cells into a single tube. 8. Count viable cells by trypan blue exclusion (APPENDIX 3B).
Labeling splenocytes for negative magnetic separation of NK cells 9. Block cells using Fc-blocking buffer at a ratio of 50 μl per 107 cells in a 50-ml conical tube for 10 min, using a platform rocker at 4°C. If cells are being used for downstream stimulations, exclude the Fc-blocking buffer and substitute isolation buffer (see recipe). Skip the pre-incubation step and directly add antibodies in isolation buffer, but anticipate a decreased cell yield.
10. During the incubation, make a master mix of the biotinylated antibodies (CD3ε, CD4, CD8, CD14, CD19, GR-1, and TER-119) at a ratio of 1 μl per 107 cells for each antibody. Titrate antibodies (UNIT 5.3) to determine optimal antibody concentrations. Do not use high concentrations of antibodies for purification, as nonspecific binding will result, causing a loss of NK cell purity.
11. Add the appropriate amount the antibody master mix to Fc-blocked splenocytes in the 50-ml tube and incubate for 20 min, using a platform rocker at 4°C. 12. Fill the 50-ml conical tube with isolation buffer and centrifuge 5 min at 585 × g, 4°C. 13. Resuspend cells in Fc blocking buffer at a ratio of 70 μl per 107 cells. 14. Add anti-biotin microbeads at a ratio of 20 μl per 107 cells and incubate for 15 min, using a platform rocker at 4°C. 15. Fill the 50-ml conical tube with isolation buffer and centrifuge 5 min at 585 × g, 4°C. 16. Resuspend the cell pellet so that there are 2 × 108 cells/ml of isolation buffer, based on cell count obtained in step 8.
Perform magnetic separation For magnetic separation of NK cells using Milenyi anti-biotin microbeads 17a. Prepare LS column by placing it in magnet and washing with 3 ml of isolation buffer. Discard the flowthrough. Pipet buffer and splenocytes onto the column through a 40-μm pre-separation filter to prevent clogging of the column. Do not let the column run dry during the magnetic separation process; therefore, do not add isolation buffer to prime column until cells are resuspended at the appropriate concentration. See UNIT 3.5A for protocols on magnetic separation.
18a. Load 0.5 ml of splenocyte suspension per column and collect flow through in a 15-ml conical tube. An LS column can hold up to 1 × 108 cells; for larger cell quantities, increase the number of LS columns appropriately.
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Table 3.22.1 Panel to Measure Purity of Isolated Murine NK Cellsa
Fluorophore
Antibody
FITC
CD3ε
PE
Ly6G (1A8)
PerCP-Cy5.5
NK1.1 (PK136)
PE-Cy7
CD19
APC
F4/80 (BM8)
AlexaFluor700
MHC class II (I-A/I-E)
APC-Cy7
CD11c
efluor450
CD11b
efluor506
Fixable cell viability marker
a The binding of the CD3ε and CD19 MAbs during the labeling process interferes with
detection during subsequent fluorophore-conjugated CD3ε and CD19 MAb labeling, so expect a lower intensity if antibody-coated cells are not efficiently removed.
19a. Wash the LS column three times, each time with 3 ml isolation buffer, into the same collection tube. 20a. Centrifuge 5 min at 585 × g, 4°C, and resuspend in a small volume. 21a. Count the cells using trypan blue exclusion (APPENDIX 3B); cells are now ready for use. Final enrichment is variable for each preparation, but generally NK purity is between 80% and 90%.
22a. Optional: If desired, remove column from magnet, place into a new 15-ml conical tube, load 5 ml of isolation buffer and, using the provided plunger, push the buffer through the column to collect cells bound to column.
For alternative magnetic separation of NK cells using anti-biotin Dynabeads 17b. Resuspend anti-biotin Dynabeads by vortexing for 30 sec and transfer desired volume of beads to a new 15-ml conical tube. Use 100 μl anti-biotin Dynabeads per 107 cells.
18b. Wash anti-biotin Dynabeads by resuspending beads in 1 ml of isolation buffer. 19b. Place the 15-ml conical tube in the magnet for 1 min and discard supernatant. 20b. Remove tube from magnet and, using isolation buffer, resuspend the beads at the original volume removed from the stock tube. 21b. Resuspend labeled cells so there are 107 cells/ml of isolation buffer. 22b. Add washed beads to the labeled cells and incubate for 30 min, using a platform rocker at 4°C. 23b. Place the 15-ml conical tube in the magnet for 2 min. 24b. Transfer the supernatant containing unbound cells to a fresh 15-ml conical tube and place it in the magnet for 2 min. Isolation of Murine Natural Killer Cells
25b. Repeat transfer of supernatant one additional time. 26b. Centrifuge 5 min at 585 × g, 4°C, and resuspend in a small volume.
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27b. Count the cells using trypan blue exclusion (APPENDIX 3B); cells are now ready for use.
Verify purity 28. To verify purity, stain cells for flow cytometry (Chapter 5). Several different panels of conjugated antibodies can be used to determine purity and phenotype, depending on the setup of the flow cytometer. Our suggested panel to measure purity is described in Table 3.22.1.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.
Fc blocking buffer [supernatant of hybridoma 2.4G2] The hybridoma supernatant is generated using a standard antibody production protocol (Yokoyama, 2008). Briefly, 2.4G2 hybridoma cells (ATCC no. HB-197) are thawed and cultured in an appropriate-sized flask in DMEM medium supplemented with 10% FBS, 10 mM HEPES, and 50 μM 2-mercaptoethanol. Cells are expanded until an adequate number of flasks can be seeded. 175-cm2 flasks are seeded at 1–2 × 105 cells/ml, and cells are allowed to grow without intervention until they begin to die (7 to 10 days). Supernatants are harvested by pelleting cells and filtering supernatant. One-tenth volume of 1 M Tris·Cl, pH 8 (APPENDIX 2A), is then added. The resultant blocking buffer then should be titered against mouse splenocytes to determine the amount of supernatant required to block staining with fluorescein-conjugated CD16 antibody as determined by flow cytometry. Supernatants are then aliquotted and stored at 4°C for up to 1 year. The blocking antibody is a rat anti-mouse product. Secondary reagents directed against rat IgG to detect other antibodies cannot be used effectively, as they will bind to the antibody in the Fc block.
Isolation buffer Phosphate-buffered saline (PBS; APPENDIX 2A) containing: 0.5% (w/v) bovine serum albumin 2 mM ethylenediaminetetraacetic acid (EDTA) Store up to 1 week at 4°C RPMI-10 medium, complete (R10) RPMI-1640 medium supplemented with: 1 × L-glutamine 1 × penicillin/streptomycin 10% (v/v) FBS (heat-inactivated 30 min at 56°C; final concentration 10% v/v) 0.1% (v/v) 2-mercaptoethanol (optional; if NK cells are to be cultured for downstream assays) Store up to 1 week at 4°C Tris/NH4 Cl lysing buffer Mix 90 ml of 0.16 M (8.3 g/liter) NH4 Cl and 10 ml of 0.17 M Tris·Cl, pH 7.65 (APPENDIX 2A); adjust to pH 7.2 with HCl. Filter sterilize as needed. Store up to 3 months at room temperature. COMMENTARY Background Information An early reported characteristic of NK cells was their morphologic appearance as large
granular lymphocytes (LGLs; Timonen et al., 1981). Earlier methods of NK cell isolation were based on differentiation from other cells
In Vitro Assays for Mouse Lymphocyte Function
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A
B 105
105
104
104 CD3ε +
43.2
69.2 0.491
0.883
103
103
102 0
102 0
1.94
0 102
103
104
0 102
105
C
5.78
103
104
105
104
105
D
105
105
104
104 0.0962
6.77e-3 0.0923
0.122
103
103
102 0
102 0
88
0 102
103
104
105
91
0 102
103
NK1.1+
Figure 3.22.1 FACS analysis of MAb-purified splenic NK cells. Representative FACS analysis of (A) unlabeled splenocytes, (B) nylon wool-enriched NK cells, (C) MAb-enriched NK cells without FcγRII/III block, and (D) MAb-enriched NK cells with FcγRII/III block. Flow cytometry data were collected on BD FACSCanto using FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star). Following exclusion of dead cells, percent NK cell enrichment was analyzed by NK1.1-PerCP-Cy5.5 and CD3ε-FITC.
Isolation of Murine Natural Killer Cells
by density separation using centrifugation with continuous (Ravnik et al., 1988) or discontinuous (Patel and Linna, 1984) Percoll gradients. Murine NK cells, however, represent a more heterogeneous population (Morelli et al., 1992) than human or rat NK cells, and the physical distinction proved to be imprecise. Although these earlier methods confirmed NK cell activity in 51 Cr-release assays against NKsensitive targets, it is now known that activated T cells can be LGLs and that small lymphocyte populations can contain NK activity. Moreover, it has been determined by flow cytometry that fewer than 40% of density-separated cells were NK1.1+ CD3ε− , particularly from spleens of C57BL/6 mice. The previous protocol employed complement-mediated lysis of contaminating cell populations. However, advances have allowed for magnetic separation of NK cells, which increases the viability of
the enriched cells and minimizes the possibility of functional alteration by complement fragments. FACS sorting is another modality for isolating NK cells by classifying splenocytes by the presence of NK1.1 and the absence of CD3ε. Although this method is both sensitive and specific, it is expensive and timeconsuming, because NK cells represent only a small fraction of splenocytes (
UNIT 3.22
Melissa A. Pak-Wittel,1 Sytse J. Piersma,1 Beatrice F. Plougastel,1 Jennifer Poursine-Laurent,1 and Wayne M. Yokoyama1,2 1
Division of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 2 Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri
ABSTRACT This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry. Curr. Protoc. Immunol.. 105:3.22.1C 2014 by John Wiley & Sons, Inc. 3.22.9. Keywords: immunology r innate immunity r cell isolation r magnetic separation
This unit describes the isolation of natural killer (NK) cells from mouse spleen. NK cells represent the third major population of lymphocytes (Yokoyama, 2013). Although NK cells were initially characterized by their tumor-killing capacity, it is now appreciated that they perform essential functions in early phases of immune response to both viral and bacterial pathogens (Horowitz et al., 2011; Vidal et al., 2011). Following activation (see UNIT 11.9B), NK cells can kill cells infected with certain intracellular pathogens, such as Cytomegalovirus, and produce cytokines that orchestrate the subsequent development of specific immunity (Scalzo et al., 2007). Moreover, the pathogen may modulate NK cell and other immune responses such that analysis of NK cell function frequently requires the study of isolated, highly purified NK cell populations. Consideration should be given to generation of interleukin 2 (IL-2)–activated NK cells [also known as lymphokineactivated killer (LAK) cells], because larger numbers are readily produced by in vitro expansion. IL-2 exposure, however, results in differences owing to activation, such as NK cell killing of a broader panel of targets, which may be inappropriate for certain experimental settings. Because NK cells constitute 2.5% of mouse splenocytes (based on NK1.1+ CD3ε− phenotype in C57BL/6 mice; Hackett et al., 1986), the isolation of purified, freshly separated NK cells poses a particular challenge. In this protocol, a method for preparing a highly purified NK cell population from mouse spleen is described. The goal is to eliminate the contaminating T cell, B cell, dendritic cell, granulocyte, and monocyte populations. This is accomplished by depleting the contaminating cells, which have been labeled with selected biotinylated monoclonal antibodies (MAbs) and magnetic beads. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, will not be functionally affected by antibody cross-linking. Additionally, no one “inert” marker has been identified that is expressed specifically on all subsets of NK cells; therefore, negative selection allows for enrichment of all NK cell subsets instead of a specific subset. Purity can then be assessed by cell surface phenotype. The MAb PK136 is directed against the Current Protocols in Immunology 3.22.1-3.22.9, April 2014 Published online April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471142735.im0322s105 C 2014 John Wiley & Sons, Inc. Copyright
BASIC PROTOCOL
In Vitro Assays for Mouse Lymphocyte Function
3.22.1 Supplement 105
cell surface molecule NK1.1 (NKR-P1C), one of the most specific markers on NK cells in C57BL/6 mice. Although a small population of NKT cells express NK1.1, NK cells should be distinguished from T cells because the NK cells are surface CD3ε− . Thus, this protocol results in an unstimulated, highly purified CD3ε− , NK1.1+ NK cell population that can be used to perform functional assays. Note that the protocol is optimized for spleen cells from C57BL/6 or C57BL/10 mice because of the detection of NK1.1+ CD3ε− NK cells in the C57BL background and due to the high prevalence of H2 congenic and genetically modified strains on this background.
Materials C57BL/6- or C57BL/10 mice Tris/NH4 Cl lysing buffer (see recipe) Complete RPMI-10 (R10) medium (see recipe) Fc blocking buffer (see recipe) or a commercially available Fc-blocking agent Isolation buffer (see recipe) 0.5 mg/ml biotinylated MAbs (eBioscience): CD3ε (145-2C11), CD8a (53-6.7), CD4 (GK1.5), CD14 (Sa2-8), CD19 (eBio1D3), TER-119, and Ly6G (GR-1) Anti-biotin microbeads (Miltenyi Biotech) Anti-biotin Dynabeads for alternative separation protocol Fluorophore-conjugated MAbs for flow cytometry: CD3ε-FITC, NK1.1-(PK136)-PerCP-Cy5.5, CD19-PE-Cy7, MHC Class II (I-A/I-E)-AlexaFluor700, CD11c-APC-Cy7, CD11b-eFluor450 (eBioscience), Ly6G (1A8)-PE and F4/80 (BM8)-AlexaFluor647 (Biolegend) Fixable cell viability marker eFluor506 for flow cytometry (eBiosciences) 70-μm cell strainer (BD Biosciences) 50- and 15-ml conical screw-cap polypropylene centrifuge tubes Beckman GS-6R swinging-bucket centrifuge with GH3.8 rotor (or equivalent) Platform rocker LS column and magnetic separator (Miltenyi Biotec; also see UNIT 3.5A) DynaMag-15 (Life Technologies) for alternate separation protocol. 40-μm pre-separation filter (Miltenyi Biotec) Additional reagents and equipment for euthanasia of mice (UNIT 1.8), removal of mouse spleen (UNIT 1.9), preparation of a cell suspension from mouse spleen (UNIT 3.1), red blood cell lysis (UNIT 3.1), use of nylon wool column for removing macrophages and dendritic cells (UNIT 3.2), counting of viable cells by trypan blue exclusion (APPENDIX 3B), antibody titration (UNIT 5.3), magnetic bead separation (UNIT 3.5A), and flow cytometry (Chapter 5) NOTE: All reagents and materials used in the preparation of these cells must be sterile if cells are to be used in culture.
Preparation of splenocyte suspension in 50-ml polypropylene centrifuge tubes 1. Euthanize mice (UNIT 1.8), and aseptically remove spleen (UNIT 1.9). 2. Prepare splenocyte suspension as described in UNIT 3.1, except use a 70-μm cell strainer, transferring cells into 50-ml polypropylene centrifuge tubes. Avoid transferring fibrous debris to the collection tube.
3. Wash cells by centrifugation 5 min at 585 × g, 4°C.
Isolation of Murine Natural Killer Cells
4. Lyse red blood cells as described in UNIT 3.1, except use 1 ml of Tris/NH4 Cl lysing buffer per spleen and, after quenching the reaction with R10 medium, filter cells through a 70-μm cell strainer into a new 50-ml conical tube. 5. Resuspend cells in R10, using 1 ml per spleen.
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6. Load and run nylon wool column as described in UNIT 3.2. Because the nylon wool column removes macrophages and dendritic cells, be careful to not overload the column. Generally, 1.5 × 108 cells for a 2-ml nylon wool column bed is ideal. For larger cell quantities, increase the number of wool columns appropriately. There is a 30% to 50% loss of total cell number on a nylon wool column.
7. Centrifuge flowthrough collected from nylon wool columns 5 min at 585 × g, 4°C. Aspirate supernatants, resuspend pellets, and, if multiple columns were used, combine cells into a single tube. 8. Count viable cells by trypan blue exclusion (APPENDIX 3B).
Labeling splenocytes for negative magnetic separation of NK cells 9. Block cells using Fc-blocking buffer at a ratio of 50 μl per 107 cells in a 50-ml conical tube for 10 min, using a platform rocker at 4°C. If cells are being used for downstream stimulations, exclude the Fc-blocking buffer and substitute isolation buffer (see recipe). Skip the pre-incubation step and directly add antibodies in isolation buffer, but anticipate a decreased cell yield.
10. During the incubation, make a master mix of the biotinylated antibodies (CD3ε, CD4, CD8, CD14, CD19, GR-1, and TER-119) at a ratio of 1 μl per 107 cells for each antibody. Titrate antibodies (UNIT 5.3) to determine optimal antibody concentrations. Do not use high concentrations of antibodies for purification, as nonspecific binding will result, causing a loss of NK cell purity.
11. Add the appropriate amount the antibody master mix to Fc-blocked splenocytes in the 50-ml tube and incubate for 20 min, using a platform rocker at 4°C. 12. Fill the 50-ml conical tube with isolation buffer and centrifuge 5 min at 585 × g, 4°C. 13. Resuspend cells in Fc blocking buffer at a ratio of 70 μl per 107 cells. 14. Add anti-biotin microbeads at a ratio of 20 μl per 107 cells and incubate for 15 min, using a platform rocker at 4°C. 15. Fill the 50-ml conical tube with isolation buffer and centrifuge 5 min at 585 × g, 4°C. 16. Resuspend the cell pellet so that there are 2 × 108 cells/ml of isolation buffer, based on cell count obtained in step 8.
Perform magnetic separation For magnetic separation of NK cells using Milenyi anti-biotin microbeads 17a. Prepare LS column by placing it in magnet and washing with 3 ml of isolation buffer. Discard the flowthrough. Pipet buffer and splenocytes onto the column through a 40-μm pre-separation filter to prevent clogging of the column. Do not let the column run dry during the magnetic separation process; therefore, do not add isolation buffer to prime column until cells are resuspended at the appropriate concentration. See UNIT 3.5A for protocols on magnetic separation.
18a. Load 0.5 ml of splenocyte suspension per column and collect flow through in a 15-ml conical tube. An LS column can hold up to 1 × 108 cells; for larger cell quantities, increase the number of LS columns appropriately.
In Vitro Assays for Mouse Lymphocyte Function
3.22.3 Current Protocols in Immunology
Supplement 105
Table 3.22.1 Panel to Measure Purity of Isolated Murine NK Cellsa
Fluorophore
Antibody
FITC
CD3ε
PE
Ly6G (1A8)
PerCP-Cy5.5
NK1.1 (PK136)
PE-Cy7
CD19
APC
F4/80 (BM8)
AlexaFluor700
MHC class II (I-A/I-E)
APC-Cy7
CD11c
efluor450
CD11b
efluor506
Fixable cell viability marker
a The binding of the CD3ε and CD19 MAbs during the labeling process interferes with
detection during subsequent fluorophore-conjugated CD3ε and CD19 MAb labeling, so expect a lower intensity if antibody-coated cells are not efficiently removed.
19a. Wash the LS column three times, each time with 3 ml isolation buffer, into the same collection tube. 20a. Centrifuge 5 min at 585 × g, 4°C, and resuspend in a small volume. 21a. Count the cells using trypan blue exclusion (APPENDIX 3B); cells are now ready for use. Final enrichment is variable for each preparation, but generally NK purity is between 80% and 90%.
22a. Optional: If desired, remove column from magnet, place into a new 15-ml conical tube, load 5 ml of isolation buffer and, using the provided plunger, push the buffer through the column to collect cells bound to column.
For alternative magnetic separation of NK cells using anti-biotin Dynabeads 17b. Resuspend anti-biotin Dynabeads by vortexing for 30 sec and transfer desired volume of beads to a new 15-ml conical tube. Use 100 μl anti-biotin Dynabeads per 107 cells.
18b. Wash anti-biotin Dynabeads by resuspending beads in 1 ml of isolation buffer. 19b. Place the 15-ml conical tube in the magnet for 1 min and discard supernatant. 20b. Remove tube from magnet and, using isolation buffer, resuspend the beads at the original volume removed from the stock tube. 21b. Resuspend labeled cells so there are 107 cells/ml of isolation buffer. 22b. Add washed beads to the labeled cells and incubate for 30 min, using a platform rocker at 4°C. 23b. Place the 15-ml conical tube in the magnet for 2 min. 24b. Transfer the supernatant containing unbound cells to a fresh 15-ml conical tube and place it in the magnet for 2 min. Isolation of Murine Natural Killer Cells
25b. Repeat transfer of supernatant one additional time. 26b. Centrifuge 5 min at 585 × g, 4°C, and resuspend in a small volume.
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27b. Count the cells using trypan blue exclusion (APPENDIX 3B); cells are now ready for use.
Verify purity 28. To verify purity, stain cells for flow cytometry (Chapter 5). Several different panels of conjugated antibodies can be used to determine purity and phenotype, depending on the setup of the flow cytometer. Our suggested panel to measure purity is described in Table 3.22.1.
REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see APPENDIX 5.
Fc blocking buffer [supernatant of hybridoma 2.4G2] The hybridoma supernatant is generated using a standard antibody production protocol (Yokoyama, 2008). Briefly, 2.4G2 hybridoma cells (ATCC no. HB-197) are thawed and cultured in an appropriate-sized flask in DMEM medium supplemented with 10% FBS, 10 mM HEPES, and 50 μM 2-mercaptoethanol. Cells are expanded until an adequate number of flasks can be seeded. 175-cm2 flasks are seeded at 1–2 × 105 cells/ml, and cells are allowed to grow without intervention until they begin to die (7 to 10 days). Supernatants are harvested by pelleting cells and filtering supernatant. One-tenth volume of 1 M Tris·Cl, pH 8 (APPENDIX 2A), is then added. The resultant blocking buffer then should be titered against mouse splenocytes to determine the amount of supernatant required to block staining with fluorescein-conjugated CD16 antibody as determined by flow cytometry. Supernatants are then aliquotted and stored at 4°C for up to 1 year. The blocking antibody is a rat anti-mouse product. Secondary reagents directed against rat IgG to detect other antibodies cannot be used effectively, as they will bind to the antibody in the Fc block.
Isolation buffer Phosphate-buffered saline (PBS; APPENDIX 2A) containing: 0.5% (w/v) bovine serum albumin 2 mM ethylenediaminetetraacetic acid (EDTA) Store up to 1 week at 4°C RPMI-10 medium, complete (R10) RPMI-1640 medium supplemented with: 1 × L-glutamine 1 × penicillin/streptomycin 10% (v/v) FBS (heat-inactivated 30 min at 56°C; final concentration 10% v/v) 0.1% (v/v) 2-mercaptoethanol (optional; if NK cells are to be cultured for downstream assays) Store up to 1 week at 4°C Tris/NH4 Cl lysing buffer Mix 90 ml of 0.16 M (8.3 g/liter) NH4 Cl and 10 ml of 0.17 M Tris·Cl, pH 7.65 (APPENDIX 2A); adjust to pH 7.2 with HCl. Filter sterilize as needed. Store up to 3 months at room temperature. COMMENTARY Background Information An early reported characteristic of NK cells was their morphologic appearance as large
granular lymphocytes (LGLs; Timonen et al., 1981). Earlier methods of NK cell isolation were based on differentiation from other cells
In Vitro Assays for Mouse Lymphocyte Function
3.22.5 Current Protocols in Immunology
Supplement 105
A
B 105
105
104
104 CD3ε +
43.2
69.2 0.491
0.883
103
103
102 0
102 0
1.94
0 102
103
104
0 102
105
C
5.78
103
104
105
104
105
D
105
105
104
104 0.0962
6.77e-3 0.0923
0.122
103
103
102 0
102 0
88
0 102
103
104
105
91
0 102
103
NK1.1+
Figure 3.22.1 FACS analysis of MAb-purified splenic NK cells. Representative FACS analysis of (A) unlabeled splenocytes, (B) nylon wool-enriched NK cells, (C) MAb-enriched NK cells without FcγRII/III block, and (D) MAb-enriched NK cells with FcγRII/III block. Flow cytometry data were collected on BD FACSCanto using FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star). Following exclusion of dead cells, percent NK cell enrichment was analyzed by NK1.1-PerCP-Cy5.5 and CD3ε-FITC.
Isolation of Murine Natural Killer Cells
by density separation using centrifugation with continuous (Ravnik et al., 1988) or discontinuous (Patel and Linna, 1984) Percoll gradients. Murine NK cells, however, represent a more heterogeneous population (Morelli et al., 1992) than human or rat NK cells, and the physical distinction proved to be imprecise. Although these earlier methods confirmed NK cell activity in 51 Cr-release assays against NKsensitive targets, it is now known that activated T cells can be LGLs and that small lymphocyte populations can contain NK activity. Moreover, it has been determined by flow cytometry that fewer than 40% of density-separated cells were NK1.1+ CD3ε− , particularly from spleens of C57BL/6 mice. The previous protocol employed complement-mediated lysis of contaminating cell populations. However, advances have allowed for magnetic separation of NK cells, which increases the viability of
the enriched cells and minimizes the possibility of functional alteration by complement fragments. FACS sorting is another modality for isolating NK cells by classifying splenocytes by the presence of NK1.1 and the absence of CD3ε. Although this method is both sensitive and specific, it is expensive and timeconsuming, because NK cells represent only a small fraction of splenocytes (
