efficient isolation of cd8α positive t cells from postnatal mice using a combined macs approach

Journal of Immunological Methods xxx (2014) xxx–xxx

JIM-11805; No of Pages 7

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Efficient isolation of CD8α positive T cells from postnatal miceusing a combined MACS approach

Anastasia Hübner a, Katja Derkowb, Anja U. Bräuer a,⁎a Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité — Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germanyb Department of Neurology, Charité — Universitätsmedizin Berlin, Berlin, Germany

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +49 30 450528405; fax:E-mail address: [email protected] (A.U. Bräu

http://dx.doi.org/10.1016/j.jim.2014.02.0060022-1759/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Hübner, A., et al., Eapproach, J. Immunol. Methods (2014), htt

a b s t r a c t

Article history:Received 7 January 2014Received in revised form 13 February 2014Accepted 13 February 2014Available online xxxx

Magnetic activated cell sorting (MACS) is a commonly used cell separation technique. MiltenyiBiotec has developed an efficient MACS protocol for isolation of CD8α+ T cells from adult micespleen. However, due to a low percentage of CD8α+ T cells in spleen of postnatal mice, MACSisolation of CD8α+ T cells results in a low purity. Our study illustrates how this problem can besolved by performing a CD45R+ B cell depletion prior to a positive selection of CD8α+ T cells.This protocol can be used when low frequencies of CD8α+ T cells are present in a tissue alsopopulated with CD45R+ B cells, and a high purity is required for downstream applications.

© 2014 Elsevier B.V. All rights reserved.

Keywords:LymphocyteSpleenDevelopmentB cell

1. Introduction

Many downstream applications require cell isolation pro-tocols that obtain high cell purity. This is especially importantwhen conducting research with tissues composed of a varietyof different cell populations, such as the spleen. Besides a highnumber of erythrocytes the spleen consists of diverse immunecell populations, including monocytes, dendritic cells, macro-phages, and B and T lymphocytes (Mebius and Kraal, 2005).The distribution of immune cells in spleens of postnatal miceis different compared to adult mice due to cell and organdevelopment. Murine T and B lymphocyte developmentappears to start as early as day 15 of gestation, increasing inpostnatal primary and secondary lymphoid organs until aplateau between 1 and 4 months in age. B cell colonization ofthe postnatal spleen occurs first, followed by colonization by Tcells (Bösing-Schneider, 1979; Holladay and Smialowicz, 2000;Velardi and Cooper, 1984). In order to isolate a certain celltype from mouse spleen a variety of protocols and techniquescurrently exist, commonly taking advantage of labeling cell type-specific membrane proteins (e.g. CD8, CD45R) with antibodies.

+49 30 450 7 528 914.er).

fficient isolation of CD8αp://dx.doi.org/10.1016/j

When using a fluorescent tagged antibody, one can perform cellenrichment and analysis by fluorescence activated cell sorting(FACS). Alternatively, themembrane protein-specific antibodiescan be tagged with super-paramagnetic beads, followed bymagnetic activated cell sorting (MACS) (Philipps et al., 2004;Pinto et al., 2013).

We chose to use MACS technique over FACS, because,workingwith rare cells, it can achieve amuch higher throughputof cells in amuch shorter time (approximately 109–1010 cells canbe sorted by MACS in about 1 h, whereas FACS is only able tosort 1% of that amount in the same time) (Givan, 2004; Philippset al., 2004).

In this study we show the significant differences of celldistributions between postnatal and adult mice and opti-mized a MACS isolation protocol for CD8α+ T cell isolationfrom postnatal mouse spleen to a final purity of N90%.

2. Materials and methods

2.1. Animals

Postnatal (P12–P15) and adult (8 weeks) male C57BL/6Nand BALB/c mice were obtained from central animal facility

positive T cells from postnatal mice using a combinedMACS.jim.2014.02.006

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2 A. Hübner et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

(FEM, Universitätsmedizin — Charité, Berlin) and kept understandard laboratory conditions (12 hour light/dark cycle;55 +/− 15% humidity; 24 +/− 2 °C room temperature (RT)and water ad libitum, enriched and grouped), in accordancewith German and European guidelines (2010/63/EU) for the useof laboratory animals. Approval of experiments was obtainedfrom the local ethics body of Berlin (LAGeSO: T0108/11).

2.2. Buffers

Erythrocyte lysis buffer: 1 mM KHCO3, 150 mM NH4Cl,0.1 mM EDTA (pH 7.43).

MACS buffer: 1 × PBS, 0.5% BSA, 2 mM EDTA (pH 7.2).

2.3. Sample preparation of murine splenocytes

MACS buffer and instruments were pre-cooled to 4 °C.Centrifugation steps took place at 300 ×g and 4 °C for 10 min.

I. Mice were sacrificed by cervical dislocation.II. Tissue was cut in halves and mashed with plunger (BD

Falcon) through moisturized nylon mesh on 50 mlconical tube (BD Falcon) (postnatal: 40 μm, adult:70 μm) and rinsed with MACS buffer until completedissociation.

III. Single cell suspension was centrifuged.IV. Aspiration of supernatant and re-suspension of cell

pellet in 1 ml erythrocytes lysis buffer followed byincubation for 5 min at RT.

V. 40 ml of MACS buffer was added into tube and cen-trifuged.

VI. Supernatant was aspirated and cell pellet wasre-suspended in 10 ml MACS-buffer.

VII. Passing of suspension through another nylon mesh(70 μm) into fresh 50 ml conical tube.

VIII. Centrifugation, aspiration of supernatant and re-suspension of cell pellet with 10 ml MACS buffer.

IX. Cell count with haemocytometer.X. A sample for the pre-isolation stain was taken at this

point by using 30,000 cells per single stain. The preparedspleen samples were immediately used for MACS.

2.4. Isolation of splenic lymphocytes using MACS

All reagents and supplies for MACS separation were pur-chased from Miltenyi Biotec, Bergisch-Gladbach, Germany.Positive selection of CD45R+ B cells from postnatal as well asfor adult BALB/c and C57BL/6Nmicewas performed according tothemanufacturer's instructions usingCD45R (B220)MicroBeads.Positive selection of CD8α+ T cells from adult BALB/c andC57BL/6N mice was performed according to themanufacturer'sinstructions using CD8a (Ly-2) MicroBeads.

For the isolation of CD8α+ T cells from spleen of BALB/c andC57BL/6N postnatal mice an adapted protocol was establishedas follows (see also Fig. 1):

A. Depletion of CD45R+ cells Labeling of entire prepared
splenocytes with CD45R (B220) MicroBeads (per-formed according to datasheet) and depletion ofCD45R+ B cells with LD column (see Fig. 1, step A).Usage of complete effluent as B cell depleted fractionfor CD8α+ T cell isolation step.
Please cite this article as: Hübner, A., et al., Efficient isolation of CD8approach, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j

B. Positive selection of CD8α+ T cells Effluent was labeled
with CD8a MicroBeads and separation followedMiltenyi protocol for CD8a (Ly-2) MicroBeads isola-tion (see Fig. 1, step B).
C. Purification of CD8α+ T cells The isolated cell fraction
was passed over a new, freshly prepared columnaccording to Miltenyi protocol to increase the purity(see Fig. 1, step C).
2.5. Flow cytometry

Cell puritywas determined by flow cytometry at a FACSCantoII (BD Biosciences, Heidelberg, Germany) both pre- and post-isolation with the respective cell marker (PE-conjugated anti-mouse CD8a, clone 53–6.7, PE-conjugated anti-mouse CD4,clone H129.19, PE-conjugated anti-mouse CD45R/B220,clone RA3-6B2, FITC-conjugated anti-mouse CD45, clone30-F11 or PE-conjugated anti-mouse CD11b, clone M1/70 allpurchased from BD Pharmingen) after exclusion of celldebris based on scatter signals and dead cells by a viability stainwith 7-AAD (eBioscience). CD16/CD32 mouse Fcγ receptorblocking, clone 2.4G2, (2.5 μg/ml, BD Pharmingen) was addedprior to staining with fluorochrome conjugated antibodies.Data analysis was performed by FACSDiva software (Version6.1.3, Becton-Dickinson).

2.6. Statistical analysis

A two-tailed nonparametric Mann–Whitney U-Test wasperformed by using the softwareGraphPadPrism (Version 5.02for Windows) to determine statistical significance. A p-valueb0.05 was considered to be statistically significant. All flowcytometric analyses were performed at least in five differentexperiments (n = 5). Data are reported as mean +/− stan-dard error of the mean (SEM).

3. Results

3.1. Mouse age dependent variations in immune cell proportions

We first determined and compared the proportions ofCD8α+, CD4+, CD45R+, CD11b+ and CD45+ cells betweenmurine postnatal and adult spleen from C57BL/6N and BALB/c mice. It was found that the percentage of CD8α+, CD4+,CD45R and CD45+ cells was lower in postnatal spleens whencompared to spleens of adult mice (Fig. 2). The differencebetween postnatal and adult mice showed significance forCD8α+ BALB/c (*p = 0.0109), CD8α+ C57BL/6N (*p =0.0112), CD4+ BALB/c (*p = 0.0117), CD4+ C57BL/6N(*p = 0.0195) and CD45R+ BALB/c (*p = 0.0119) cells.However, CD45R cells isolated from C57BL/6N spleen showedno significant difference. Notably, the reduced percentage ofCD8α+ T cells in postnatal (~3%) compared to adult mice(~16.6%) was prominent and emerged as a problem whenapplying the standard MACS separation protocol. Further-more, the percentage of CD11b+ cells in the spleen wasdetermined between postnatal (10.3%) and adult (12.8%)mice (Fig. 2D). However, this difference was non-significant.To determine the starting percentage of leukocytes, a stainingwith CD45+ conjugated fluorescent antibodies was per-formed on splenic cells. As shown in Fig. 2E, the amount of

α positive T cells from postnatal mice using a combinedMACS.jim.2014.02.006


A) B) C)

Fig. 1. Scheme of the altered protocol for purification of CD8α positive T cells from postnatal murine splenocytes.

Fig. 2. Flow cytometric characterization of BALB/c and C57BL/6N immune cell populations in postnatal and adult murine splenocytes. These cells were isolatedfrom postnatal or adult mice, and then measured by flow cytometry for expression of either A. CD8α, B. CD4, C. CD45R, D. CD11b, or E.CD45. All flow cytometricanalyses were performed at least in five different experiments (n = 5). Exclusion of dead cells and debris. Data are reported as mean +/− standard error of themean (SEM), *p b 0.05, Mann-Whitney U-test.

3A. Hübner et al. / Journal of Immunological Methods xxx (2014) xxx–xxx

Please cite this article as: Hübner, A., et al., Efficient isolation of CD8α positive T cells from postnatal mice using a combinedMACSapproach, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.02.006



leukocytes following lysis of erythrocytes is about 91% inadult mice. In contrast, we observed 54% leukocytes in postnatalmice (***p = 0.0007).

Our investigations showed furthermore that cell percent-ages within the spleen can also slightly differ between BALB/cand C57BL/6N strains (Fig. 2). Considering CD8α+ and CD45R+

cells, the difference was non-significant, but the populations ofCD4+ cells were significantly higher (*p = 0.02) in BALB/c thanin C57BL/6 adult mice (Fig. 2B). The cell proportions of CD11b+

and CD45+ cells showed no significant difference between thestrains (data not shown).

3.2. Isolation of CD8α+ T cells by altered MACS protocol yieldspurities over 90% for postnatal mice

Neither by using positive selection with CD8a MicroBeadsnor by depleting all non-wanted cells by a specific CD8α Tcell isolation kit could we gain sufficiently high purities ofCD8α+ T cells in postnatal mice. Only by our adapted MACSprotocol (Fig. 1; steps A–C), were we able to increase purityup to 90%. After depletion of CD45R+ B cells (Fig. 1; step A), weraised the amount of CD8α+ T cells from 3% to 7% (data notshown). Subsequently, by the positive selection of CD8α+ Tcells (Fig. 1; step B), purities of 57.8% for C57BL/6N and 73.2%for BALB/c mice were obtained (positive cell fraction 1, Fig. 3Aand B). Following purification of the CD8α positive cell fractionover a second column (Fig. 1; step C), we finally gained puritiesof 92.3% (C57BL/6N) and 96.0% (BALB/c) (Fig. 3A and B) forisolation of CD8α+ T cells from spleen of postnatal mice.

Isolation of CD8α+ T cells from adult mice spleens requiredpassing over another column for efficient purification of the cellfraction post-sort according toMiltenyi standard protocol. Withthe standard procedure a yield of 82.2% CD8α+ T cells fromBALB/c (CD8α positive fraction 1, Fig. 3A) and 78.1% CD8α+ Tcells from C57BL/6N (CD8α positive fraction 1, Fig. 3B) micewas obtained. The second CD8α+ fraction yielded a purity of93.3% for BALB/c (Fig. 3A) and 86.9% for C57BL/6N (Fig. 3B)after being sorted over a new column. These purities arecommon for adult mice according to Hamelmann et al. (1996)and Hoerauf et al. (1996).

In comparison, the number of CD45R+ B cells is justmarginally lower in postnatal than in adult mice (Fig. 2C, 4Aand B). The sort of these cells both in postnatal and adultmice was comparable and within a yield expected from thestandardized MACS protocol with CD45R MicroBeads. Wereached purities in postnatal stages of 90.9% in BALB/c(Fig. 4A), 92.4% in C57BL/6N (Fig. 4B) and in adult mice of94.5% in BALB/c (Fig. 4A) and 94.2% in C57BL/6N (Fig. 4B).

4. Discussion

The reason for applying an altered MACS protocol is basedon the lowpercentage of CD8α+T cells in postnatalmice of ourinvestigated strains C57BL/6 and BALB/c. CD8α is an isoformof the CD8 antigen, a cell surface glycoprotein. It is found on

Fig. 3. Flow cytometric characterization of A. BALB/c and B. C57BL/6N immune cell pisolated from postnatal and adult BALB/c and C57BL/6N mice, postnatal cells werprotocol, measured by flow cytometry for expression of CD8α before and after ismean +/− standard error of the mean (SEM).

Please cite this article as: Hübner, A., et al., Efficient isolation of CD8αapproach, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j

most cytotoxic T lymphocytes and mediates efficient cell–cellinteractions. Although CD8α is also expressed on a subset ofdendritic cells (Kamath et al., 2000), it is generally accepted as aT-cell marker. However, following CD8α positive selectionwe were only able to obtain a purity of approximately 66%.According to Miltenyi Biotec the CD8a MicroBeads for positiveselection are designed for efficient isolation (above 90%) whenthe cell population comprises above 7% and up to 15% of totalcells (S. Heidmeier (technical support, Miltenyi Biotec GmbH),personal communication). This corresponds with our findingsfrom a mean level of 17.5% CD8α+ T cells for adult BALB/cand 15.8% CD8α+ T cells for adult C57BL/6N mice spleen. Incontrast CD8α+ T cells in postnatal micewere found in amuchlower frequency (~3%). Therefore it was essential to enrichthe CD8α+ T cell population in order to conduct any otherdownstream applications. Our first experiments showed that adepletion of the large CD45R+ B cell fraction (31.1% in C57BL6/N and 28.0% in BALB/c) raised the percentage of CD8α+ T cellsfrom 3% to 7% (data not shown). CD45R (B220) is a 220 kDaprotein isoform of CD45, a common immune cell surfacemembrane protein. In general, CD45 functions as a marker forleukocytes and its expression is related to different develop-mental stages (Abdul-Salam et al., 2001). For example, CD45negative cells can be found at early postnatal stages already.The CD45Rmonoclonal antibody used in this study is known tobe a pan-specific B-cell marker. However, it is also expressedon activated T cells, subsets of dendritic cells, as well as otherantigen presenting cells (Marvel and Mayer, 1988). Early inpostnatal development one can expect a low frequency ofactivated CD8α+T cells in the spleen; thereforewedeterminedthat CD45R may serve as a suitable marker for cell depletionusing theMACS protocol outlined byMiltenyi Biotec.We foundthat CD45R+ B cell depletion of postnatal mouse splenic cells,followed by isolation of CD8α+ T cells increased CD8α+ cellpurity by approximately 30% when compared to performingthe CD8α positive selection alone. We also applied a differentgating strategy for flow cytometry than was outlined by aMiltenyi Biotec representative. We only excluded cell debrisand dead cells without any further restrictions. The Miltenyigating strategy involved gating on the CD45+ leukocyte fraction,followed by gating on the CD3+ T cell fraction. CD8α+ T cellscan be found within the CD3+ fraction at a purity of nearly100% in 8 week old C57BL/6N mice (personal communication,S. Heidmeier, technical support, Miltenyi Biotec GmbH). Wepreferred to exclude less cell populations to increase theCD8α+ T cell purity in our samples.

In this studyweprovide an optimizedprotocol for isolation ofCD8α+ T cells from postnatal mouse spleen. This will be usefulfor downstream applications where high cell purity is essential.For example, gene and protein expression techniques suchas quantitative PCR or mass spectrometry respectively, oftendemand highly pure cell preparations for accurate interpretationof data. Similarly, cell culture techniques also require high cellpurity, as contamination of other cell populations can influencetarget cell growth, differentiation and gene expression.

roportions between postnatal and adult murine splenocytes. These cells weree sorted according to Fig. 1 and adult cells to standard MicroBeads (CD8a)olation, exclusion of dead cells and cell debris, n = 5. Data are reported as




Acknowledgments

Rike Dannenberg and Jan Csupor are acknowledged fortheir excellent technical assistance. Dipl. Biol. Ulrike Schließerand Dr. Sandra Gültner (Miltenyi Biotec) are acknowledged fortheir collaboration.Dr. Christine Brandt, Prof. Dr. Birgit Sawitzkiand M.Sc. Daniel J. Neuman are acknowledged for their helpfuldiscussions. M.Sc. Daniel J. Neuman is further acknowledgedfor editing the manuscript. This work was supported by theDFG BR 2345/1-1 to A.U.B. and the Sonnenfeld-Stiftung forsponsoring technical equipment for A.U.B.

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Please cite this article as: Hübner, A., et al., Efficient isolation of CD8αapproach, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j

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efficient isolation of cd8α positive t cells from postnatal mice using a combined macs approach

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