Development and migration of plasma cells in the mouse lymph node.

Animals, Immunization, Transfer For adoptive transfer experiments, wild-type C57BL/6, B6.SJL (CD45.1 + ), CFP + , and dsRed + mice (Jackson Laboratories) between 7–12 weeks of age were used with protocols approved by Institutional Animal Care and Use Committee. CD45.1 + hosts were immunized with an intrapertioneal (IP) immunization of 50µg/mouse of OVA (Sigma) emulsified in Alum (Pierce), to enrich T-cell help two weeks prior to cell transfer. B1.8 high Blimp-1-YFP mice (with or without CFP + transgene) were used for donor cells. Splenic naive B-cells were purified by CD43-depletion using MACS® beads (Miltenyi Biotec) and transferred intravenously (IV) to hosts (5–10×10 6 /mouse). After 24 hours, hosts received a subcutaneous (S/C) boost in the footpads with 100 µg of NP(15)-OVA (Biosearch). Lymphocytes were harvested and analyzed 3 to 10 days later by flow cytometry on a LSR II (BD Bioscences). For imaging experiments, hosts were imaged on day 4, 7 and 10 post-boost with NP-OVA. One day prior to imaging, naïve control B-cells purified from CFP + B6 mice were adoptively transferred into the mouse recipient as motility controls. To label medullary cord macrophages, 1pmole of QTracker© 705 non-targeted quantum dot (Invitrogen) was injected in the hind footpad. In addition, 2.5µg of NP(11)-Tomato or NP- Phycoerythrin (PE) (Biosearch) was injected in the hind footpad to label the light zone of the germinal center structures. NP-tomato was made in house, as a tomato-GST construct, expressed in e coli , purified by Glutathione column (Amersham), and conjugated with NP-SE (Biosearch). For in vivo experiments, Pertussis toxin (Ptx, 400 µg/kg Calbiochem) was administered IV, FTY720 (1 mg/kg) was delivered IP 12–24h prior to imaging ( Huang et al., 2007 ). In vitro Imaging and Transwell Blimp-YFP + naive B cells were purified and stimulated with LPS (25ng/mL), IL-4 (5ng/mL), IL-5 (15ng/mL), and BAFF (10ng/mL) for 3–4 days. Naive dsRed + B cells were purified and incubated in RPMI+10% FBS overnight before imaging or transwell experiments. For transwell experiments, cells were incubated in upper chamber of 5µm Costar transwells for two hours with CXCL12 (1µg/mL, R&D Systems) and were counted on a FACScalibur. For imaging, cells were imaged in Lab-Tek ™ chamber slides systems (Nunc) using an adapted protocol as kindly provided from the laboratory of Ronen Alon at the Weizmann Institute of Science in Rehovot, IL. Chemokines were spotted on clean glass surface for 2h at 37°C, washed, and then spotted with 5 µL of transmembrane ICAM-1 at 400 molecules/µm 2 for 1hr at room temperature. The glass surface was blocked with 10% bovine serum albumin (BSA) or 10% Fatty acid free (FAF)-BSA for 1hr, washed, and then used for imaging. Prior to imaging, cells were incubated in RPMI+1% BSA (Regular or FAF, Sigma) with or without Ptx (50ng/mL) for two hours. Cells were resuspended in HBSS supplemented with 10mM HEPES, 2mg/mL BSA (FAF or Regular), and imaged at 37°C using a Zeiss LSM710 using standard confocal settings for YFP and dsRed fluorophores.

TPLSM Imaging and Analysis

To prepare for imaging, mice were anesthetized using KXA (Ketamine, Xyalzine, Acepromazine) solution (4µl/g). We exposed the popliteal lymph node by shaving the leg, dissecting the skin and fatpad in an aseptic manner. A metal stabilization plate and coverslip were used to immobilize the lymph node. The mouse was comforted with regular injections of KXA, temperature control stage, and nose-cone with O 2 supplement. For intravital TPLSM, an upright BioRad microscope was used as before ( Schwickert et al., 2007 ) using a 40X Nikon objective and also repeated on a Zeiss LSM-710 inverted microscope with Mai-Tai Sapphire Laser for two-photon excitation using a 25X objective. Time lapse z-stacks were taken at 30 sec intervals on either system, with similar measurements for cell speeds and migratory behavior. Tiled 3D stacks of the lymph node were collected on the Zeiss system. Collected imaging series were analyzed for cell position and dynamics using Volocity 4.2 (Improvision), Excel 2003 (Microsoft) and GraphPad 4.0 (Prism). Movies were annotated using Adobe Affect Effects. Cell migrations were tracked automatically using Volocity, annotated for region and pruned manually. Only continuous tracks that persisted at least 6 minutes in the field were used for analysis. For cell distributions within a lymph node, cell bodies were identified by RGB thresholding to cells. Rather than segmenting individual cells, which frequently clustered particularly in the medullary cords, total cell pixel volumes were determined, subdivided by region in the lymph node, and presented as a percentage found in the total lymph node. Both sides of the lymph node were totaled for each measurement and repeated for 4–6 lymph nodes per condition. For MSD measurements, sampling was rounded to the nearest minute. The initial 30 minutes of the MSD plots were fit to a 3D persistent random walk ( Equation 1 ) as (Equation 1) MSD ( t ) = 3 S 2 [ Pt − P 2 ( 1 − e − t / P ) ] described ( Zygourakis, 1996 ) using non-linear least squares (Origin 7.0) and solved for persistence time (P) using the average constant speed from Figure 3B as speed (S). Cell Staining, Flow Cytometry Flow cytometry measurements were conducted on a BD LSR II and analyzed using FlowJo 7.2. Cell populations were stained using BD Pharmagen antibodies unless otherwise stated: CD19, B220, CD138, CXCR5, CXCR4, Fas, GL7, and CD45.1.

Electron Microscopy Preparation and Imaging

Cells were washed with serum-free media or appropriate buffer then fixed with a modified Karmovsky's fix of 2.5% glutaraldehyde, 4% paraformaldehyde and 0.02% picric acid in 0.1M sodium caocdylate buffer at pH 7.2. Following a secondary fixation in 1% osmium tetroxide, 1.5% potassium ferricyanide, samples were dehydrated through a graded ethanol series, cleared with acetonitrile then infiltrated and embedded in an epon analog resin. Ultrathin sections were cut using a Diatome diamond knife (Diatome) on a Leica Ultracut S ultramicrotome (Leica). Sections were collected on copper grids, further contrasted with lead citrate, and viewed on a JSM 100 CX-II electron microscope (JEOL, USA, Inc., Peabody, MA) operated at 80 kV. Images were recorded on Kodak 4489 Electron Image film then digitized on an Epson Expression1600 Pro scanner.

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