Name: retroviral overexpression of cxcr4 on murine b-1a cells and adoptive transfer for targeted b-1a cell migration to the bone marrow and igm production
Uploaded: 2020-05-31T13:00:00
Description: Watch this Scientific Journal Video about Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production at JoVE.c

This work was supported by 1R01 HL107490, 1R01 HL136098, Project 3 of P01 HL055798, P01 HL136275-01 (C.A. McNamara), and R01GM100776 (T.P. Bender). A. Upadhye was supported by American Heart Association Pre-doctoral fellowship 16PRE30300002 and 5T32AI007496-20. We thank Joanne Lannigan, Mike Solga, and Claude Chew from the University of Virginia Flow Cytometry Core for their excellent technical assistance.

All animal protocols were approved by the Animal Care and Use Committee at the University of Virginia.
1. Magnetic separation and enrichment of peritoneal B-1 cells
<ol>
	<li>Euthanize a 12&#8722;14-week-old, male, CD45.1+ApoE-/- mouse using CO2.</li>
	<li>Make a superficial cut in the abdomen using straight surgical scissors and peel back skin using curved scissors to expose the peritoneal wall. Flush peritoneal cavity with 10 mL of 37 &#176;C RPMI-1640 medi...

<ol>
	<li>Baumgarth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+Cell+Heterogeneity+and+the+Regulation+of+Natural+and+Antigen-Induced+IgM+Production.">B-1 Cell Heterogeneity and the Regulation of Natural and Antigen-Induced IgM Production.</a> Frontiers in Immunology. 7, 324 (2016).</li><li>Holodick, N. E., Vizconde, T., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1a+cell+diversity:+nontemplated+addition+in+B-1a+cell+Ig+is+determined+by+progenitor+population+and+developmental+location.">B-1a cell diversity: nontemplated addition in B-1a cell Ig is determined by progenitor population and developmental location.</a> Journal of Immunology. 192 (5), 2432-2441 (2014).</li><li>Prohaska, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massively+Parallel+Sequencing+of+Peritoneal+and+Splenic+B+Cell+Repertoires+Highlights+Unique+Properties+of+B-1+Cell+Antibodies.">Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies.</a> Journal of Immunology. 200 (5), 1702-1717 (2018).</li><li>Upadhye, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversification+and+CXCR4-Dependent+Establishment+of+the+Bone+Marrow+B-1a+Cell+Pool+Governs+Atheroprotective+IgM+Production+Linked+to+Human+Coronary+Atherosclerosis.">Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.</a> Circulation Research. 125 (10), 55-70 (2019).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+define+murine+B+cell+lineage+immunoglobulin+heavy+chain+(IgH)+repertoires.">Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires.</a> Elife. 4, 09083 (2015).</li><li>Choi, Y. S., Dieter, J. A., Rothaeusler, K., Luo, Z., Baumgarth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+cells+in+the+bone+marrow+are+a+significant+source+of+natural+IgM.">B-1 cells in the bone marrow are a significant source of natural IgM.</a> European Journal of immunology. 42 (1), 120-129 (2012).</li><li>Holodick, N. E., Tumang, J. R., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+secretion+by+B1+cells:+Differential+intensity+and+IRF4-dependence+of+spontaneous+IgM+secretion+by+peritoneal+and+splenic+B1+cells.">Immunoglobulin secretion by B1 cells: Differential intensity and IRF4-dependence of spontaneous IgM secretion by peritoneal and splenic B1 cells.</a> European Journal of Immunology. 40 (11), 3007-3016 (2010).</li><li>Moon, H., Lee, J. G., Shin, S. H., Kim, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS-induced+migration+of+peritoneal+B-1+cells+is+associated+with+upregulation+of+CXCR4+and+increased+migratory+sensitivity+to+CXCL12.">LPS-induced migration of peritoneal B-1 cells is associated with upregulation of CXCR4 and increased migratory sensitivity to CXCL12.</a> Journal of Korean Medical Science. 27 (1), 27-35 (2012).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+plasma+cell+alloantigen+1+defines+layered+development+of+B-1a+B-cell+subsets+with+distinct+innate-like+functions.">Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (49), 20077-20082 (2012).</li><li>Yang, Y., Tung, J. W., Ghosn, E. E., Herzenberg, L. A., Herzenberg, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+and+differentiation+of+natural+antibody-producing+cells+in+mouse+spleen.">Division and differentiation of natural antibody-producing cells in mouse spleen.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (11), 4542-4546 (2007).</li><li>Tsiantoulas, D., Gruber, S., Binder, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B-1+cell+immunoglobulin+directed+against+oxidation-specific+epitopes.">B-1 cell immunoglobulin directed against oxidation-specific epitopes.</a> Frontiers in Immunology. 3, 415 (2012).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>McMahon, S. B., Norvell, A., Levine, K. J., Monroe, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+of+murine+B+lymphocyte+blasts+as+a+method+for+examining+gene+regulation+in+primary+B+cells.">Transient transfection of murine B lymphocyte blasts as a method for examining gene regulation in primary B cells.</a> Journal of Immunological Methods. 179 (2), 251-259 (1995).</li><li>Lin, K. I., Calame, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+genes+into+primary+murine+splenic+B+cells+using+retrovirus+vectors.">Introduction of genes into primary murine splenic B cells using retrovirus vectors.</a> Methods in Molecular Biology. 271, 139-148 (2004).</li><li>Moghimi, B., Zolotukhin, I., Sack, B. K., Herzog, R. W., Cao, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Efficiency+Ex+Vivo+Gene+Transfer+to+Primary+Murine+B+Cells+Using+Plasmid+or+Viral+Vectors.">High Efficiency Ex Vivo Gene Transfer to Primary Murine B Cells Using Plasmid or Viral Vectors.</a> Journal of Genetic Syndromes and Gene Therapy. 2 (103), 1000103 (2011).</li><li>DeKoter, R. P., Lee, H. J., Singh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PU.1+regulates+expression+of+the+interleukin-7+receptor+in+lymphoid+progenitors.">PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors.</a> Immunity. 16 (2), 297-309 (2002).</li><li>Holodick, N. E., Vizconde, T., Hopkins, T. J., Rothstein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Decline+in+Natural+IgM+Function:+Diversification+and+Selection+of+the+B-1a+Cell+Pool+with+Age.">Age-Related Decline in Natural IgM Function: Diversification and Selection of the B-1a Cell Pool with Age.</a> The Journal of Immunology. 196 (10), 4348-4357 (2016).</li><li>Laurie, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+and+efficient+expression+in+mouse+and+human+B+cells+by+a+novel+hybrid+immunoglobulin+promoter+in+a+lentiviral+vector.">Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.</a> Gene Therapy. 14 (23), 1623-1631 (2007).</li><li>Warncke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vitro+transduction+of+naive+murine+B+cells+with+lentiviral+vectors.">Efficient in vitro transduction of naive murine B cells with lentiviral vectors.</a> Biochemical and Biophysical Research Communications. 318 (3), 673-679 (2004).</li><li>Moon, B. G., Takaki, S., Miyake, K., Takatsu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+IL-5+for+mature+B-1+cells+in+homeostatic+proliferation,+cell+survival,+and+Ig+production.">The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production.</a> Journal of Immunology. 172 (10), 6020-6029 (2004).</li><li>Takatsu, K., Kouro, T., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+5+in+the+link+between+the+innate+and+acquired+immune+response.">Interleukin 5 in the link between the innate and acquired immune response.</a> Advances in Immunology. 101, 191-236 (2009).</li><li>Baumgarth, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+double+life+of+a+B-1+cell:+self-reactivity+selects+for+protective+effector+functions.">The double life of a B-1 cell: self-reactivity selects for protective effector functions.</a> Nature Reviews Immunology. 11 (1), 34-46 (2011).</li></ol>

The method provided here enables stable and relatively efficient primary B-1a cell gene delivery, in vivo adoptive transfer, and identification and localization of injected cells. Cells were able to be detected 17 weeks post-cell transfer and retained increased CXCR4 expression. Retrovirus-mediated delivery yielded 30-40% transduction efficiency of primary murine B-1a cells with minimal impact on cell viability in our hands (Figure 4e). This is in line with results from a previous study by M...

Recent studies have demonstrated considerable immune cell, and specifically B cell, phenotypic and functional heterogeneity depending on cell localization1,2,3,4,5. B-1a cells are one such population with heterogeneous capacity to produce protective IgM antibodies; bone marrow B-1a cells secrete IgM constitutively and contribute significantly to plasma IgM titers6, while peritoneal B-1a cells have low-level IgM secretion at homeostasis and instead can be activated through innate toll-like receptor (TLR) or cytokine-mediated signaling to rapidly proliferate, migrate, and secrete IgM7,8,9,10. B-1a cell IgM antibodies recognize oxidation-specific epitopes (OSE) that are present on pathogens, apoptotic cells, and oxidized LDL, and IgM binding to OSE can prevent inflammatory downstream signaling in diseases like atherosclerosis11. Therefore, strategies to increase IgM production via increasing peritoneal B-1a cell migration to sites like the bone marrow may be therapeutically useful. However, it is important for such strategies to be targeted and cell-type specific, as off-target effects may negatively impact immune function or health.
Here we describe a method for targeted and long-term overexpression of CXCR4 in primary murine B-1a cells and subsequent adoptive transfer to visualize cell migration and functional IgM antibody production (Figure 1). Genetic manipulation of primary B cells is limited by low transfection efficiencies compared to transfection of transformed cell lines. However, as transformed cell lines can significantly deviate from primary cells12,13, the use of primary cells is likely to provide results that more closely align to normal physiology. Several techniques have been described for gene transfer in primary murine B cells, including retroviral transduction, adenoviral transduction, lipofection, or electroporation-based transfection, which have varying levels of efficiency, transience, and impact on cell health13,14,15. The following method utilized retroviral transduction as it yielded adequate gene transfer efficiency of &#62;30% while minimally impacting cell viability. The CXCR4-expressing retrovirus was generated using the previously described retroviral construct murine stem cell virus-internal ribosomal entry site-green fluorescent protein (MSCV-IRES-GFP; MigR1)16, into which the mouse CXCR4 gene was sub-cloned4. MigR1 (control(Ctl)-GFP) and CXCR4-GFP retroviral particles were generated using calcium phosphate transfection as described in previously published protocols4,14.
Successfully transduced B-1a cells were then intravenously transferred into lymphocyte-deficient Rag1-/- mice. Both donor and recipient mice additionally contained knockout of the apolipoprotein E (ApoE) gene, which results in increased OSE accumulation and atherosclerosis, thereby providing a model for in vivo B-1 cell activation and IgM production. Moreover, donor and recipient mice differed in CD45 allotype; donor B-1 cells came from CD45.1+ ApoE-/- mice and were transferred into Rag1-/- CD45.2+ ApoE-/- recipients. This allowed differentiation of donor CD45.1 from recipient CD45.2 B cells post-transfer without the need to additionally stain for B cell markers during flow cytometry analysis. The results provided here demonstrate that targeted CXCR4 overexpression on B-1a cells associates with increased ability of B-1a cells to migrate to the bone marrow, which associates with increased plasma anti-OSE IgM. We additionally provide a method for the enrichment of peritoneal B-1 cells through negative selection and demonstrate the requirement of B-1 cell activation for efficient transduction. This method can be adapted for other retroviral constructs to study the effect of protein overexpression on B-1a cell migration, phenotype, or function. Moreover, the use of CD45.1 versus CD45.2 allotype distinction could theoretically allow transfer into other immune-sufficient murine models containing endogenous B cells.

<table>
	<tbody>
		<tr>
			<td>70 micron filter caps</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-biotin microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD16/CD32, or Fc block</td>
			<td>Life Technologies</td>
			<td>MFCR00</td>
			<td></td>
		</tr>
		<tr>
			<td>B220 APC</td>
			<td>eBioscience</td>
			<td>17-0452-83</td>
			<td>Clone: RA3-6B2</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 APCef780</td>
			<td>eBioscience</td>
			<td>47-0193-82</td>
			<td>Clone: eBio1D3</td>
		</tr>
		<tr>
			<td>CD23 biotin</td>
			<td>eBioscience</td>
			<td>13-0232-81</td>
			<td>Clone: B3B4</td>
		</tr>
		<tr>
			<td>CD23 PECy7</td>
			<td>eBioscience</td>
			<td>25-0232-82</td>
			<td>Clone: B3B4</td>
		</tr>
		<tr>
			<td>CD3e biotin</td>
			<td>eBioscience</td>
			<td>13-0033-85</td>
			<td>Clone: eBio500A2</td>
		</tr>
		<tr>
			<td>CD45.1 ApoE-/- mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in house</td>
		</tr>
		<tr>
			<td>CD45.1 PerCP-Cy5.5</td>
			<td>BD Biosciences</td>
			<td>560580</td>
			<td>Clone: A20</td>
		</tr>
		<tr>
			<td>CD45.2 BV421</td>
			<td>BD Biosciences</td>
			<td>562895</td>
			<td>Clone: 104</td>
		</tr>
		<tr>
			<td>CD45.2 Rag1-/- ApoE-/- mice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Bred in house</td>
		</tr>
		<tr>
			<td>CD5 PE</td>
			<td>eBioscience</td>
			<td>12-0051-83</td>
			<td>Clone: 53-7.3</td>
		</tr>
		<tr>
			<td>Ctl-GFP retrovirus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generated in house using GFP-expressing retroviral plasmid MigR1 provided by Dr. T.P. Bender</td>
		</tr>
		<tr>
			<td>CXCR4 APC</td>
			<td>eBioscience</td>
			<td>17-9991-82</td>
			<td>Clone: 2B11</td>
		</tr>
		<tr>
			<td>CXCR4-GFP retrovirus</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Generated in house by cloning mouse CXCR4 into MigR1 retroviral plasmid</td>
		</tr>
		<tr>
			<td>F4/80 biotin</td>
			<td>Life Technologies</td>
			<td>MF48015</td>
			<td>Clone: BM8</td>
		</tr>
		<tr>
			<td>Flowjo Software v. 9.9.6</td>
			<td>Treestar Inc.</td>
			<td>License required</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Gr-1 biotin</td>
			<td>eBioscience</td>
			<td>13-5931-82</td>
			<td>Clone: RB6-8C5</td>
		</tr>
		<tr>
			<td>heat-inactivated fetal bovine serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>IgM PECF594</td>
			<td>BD Biosciences</td>
			<td>562565</td>
			<td>Clone: R6-60.2</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD Biosciences</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Henry Schein Animal Health</td>
			<td>029405</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Yellow</td>
			<td>Life Technologies</td>
			<td>L34968</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>NK1.1 biotin</td>
			<td>BD Biosciences</td>
			<td>553163</td>
			<td>Clone: PK136</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>ODN 1668</td>
			<td>InvivoGen</td>
			<td>tlrl-1668</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119 biotin</td>
			<td>eBioscience</td>
			<td>13-5921-82</td>
			<td>Clone: Ter119</td>
		</tr>
	</tbody>
</table>

retroviral overexpression of cxcr4 on murine b-1a cells and adoptive transfer for targeted b-1a cell migration to the bone marrow and igm production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

An overview of the protocol is given in Figure 1. Figure 2 displays enrichment of peritoneal B-1a cells after magnetic depletion of other peritoneal cell types. Live singlet cells in the post-depletion fraction have a greater proportion of CD19+ B cells compared to F4/80+ macrophages, lack CD5hi CD19- T cells, and contain an increased frequency of CD19+ CD5mid B-1a cells compared to the pre-depletion fractio...

Watch this Scientific Journal Video about Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production at JoVE.c

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

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Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...