This study was supported by a research grant from the Ministry of Science and Technology of the Executive Yuan of Taiwan (NSC99-2320-B-002-011). I would like to acknowledge the excellent service provided by the Flow Cytometric Analyzing and Sorting Core of the First Core Laboratory in College of Medicine of National Taiwan University.

Human peripheral blood must be obtained from healthy donors under informed consent, and the use of blood samples must conform to the approved guidelines established by individual institutional review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cytometry (Figure 1) and ELISpot assays (Figure 3) was approved by the Internal Review Board of National Taiwan University Hospital (protocol number 201307019RINB).
1. Isolation and Purification of Human Peripheral Blood B Cells
<ol>
	<li>Draw ~ 10 mL of blood from the median cubital vein (in the cubital fossa anterior to the elbow) into a 15-mL tube containing K2EDTA (1.5 to 2.0 mg/mL blood) and immediately invert the tube several times to prevent clot formation.</li>
	<li>Add 35 mL of autoclaved (121 &#176;C, 15 min) red blood cell (RBC) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA; pH 7.4) to the tube containing the fresh blood sample (&#8805; 3:1 vol/vol) and incubate at room temperature (RT) for no longer than 5 min. 
	NOTE: The appearance of light transmission through the tube indicates the completion of RBC lysis.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Ensure that the pellet is white in color.</li>
	<li>Resuspend the pellet with 10 mL of autoclaved phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4; pH 7.4) and centrifuge as in step 1.3.</li>
	<li>Discard the supernatant, resuspend the pellet with 10 mL of RPMI 1640 medium (supplements: 10% fetal calf serum, 100 U/mL penicillin/streptomycin, 0.25 &#181;g/mL amphotericin B, and 2 mM L-glutamine), and then plate the cells into a 10-cm culture dish (10 mL of blood per dish). Place the dish in a 37 &#176;C incubator with 5% CO2 for 30 min.</li>
	<li>Gently swirl the culture dish a few times and place the culture medium (suspension cells) into 15-mL plastic conical tubes. Discard the adherent cells (mostly macrophages) on the culture dishes.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet with 1 mL of RPMI 1640 medium. Count the cell number with a hemocytometer or an automated cell counter. 
	NOTE: The viability of isolated leukocytes is normally greater than 90% by trypan blue exclusion.</li>
	<li>Centrifuge as in step 1.7 and resuspend the cells in ~ 200 &#181;L of cold PBS buffer (0.5% bovine serum albumin (BSA) and 2 mM EDTA) at the concentration of 5 - 10 x 106 cells/mL.</li>
	<li>Add 5 &#181;L of biotinylated anti-human Ab cocktail specific to blood cells (for the negative selection of B cells) per 106 cells and incubate on ice for 30 min21. 
	NOTE: The anti-human Ab cocktail should at least include Abs specific to CD2 (or CD3), CD14, and CD16.</li>
	<li>Add a 10-fold excess volume of sterile PBS to the cells, centrifuge at 600 x g for 5 min, and discard the supernatant.</li>
	<li>Add equal amounts of streptavidin-conjugated microbeads (5 &#181;L per 106 cells) to the pellet and mix thoroughly.</li>
	<li>Incubate on ice for 30 min in a 15-mL plastic conical tube.</li>
	<li>Add 2 mL of PBS buffer into the tube.</li>
	<li>Place the tube into a magnetic stand and incubate at RT for 8 min. The brown microbeads will gradually attach to the tube wall next to the magnet22.</li>
	<li>With the tube remaining in the magnetic stand, carefully transfer the supernatant into a new sterile 15-mL tube22.</li>
	<li>Repeat steps 1.14 to 1.16, and combine the two supernatants that contain the untouched B cells. Discard the microbeads.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Discard the supernatant.</li>
	<li>Resuspend the pellet in RPMI 1640 medium for downstream experiments. 
	&#8203;NOTE: Typically, 1 - 5 x 105 B cells with a purity greater than 95% from 10 mL of peripheral blood can be isolated23.</li>
</ol>
2. Purification and Separation of Memory and Na&#239;ve B Cells from Isolated B Cells
<ol>
	<li>Use cells purified from step 1 and determine the cell number using a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells in 100 &#181;L of cold PBS buffer after centrifugation at 600 x g and RT for 5 min.</li>
	<li>Add 1 - 2 &#181;g of biotinylated CD27 mAb per 106 cells and incubate on ice for 30 min.</li>
	<li>Add 10 mL of PBS buffer to the tube and centrifuge at 600 x g for 5 min.</li>
	<li>Discard the supernatant and resuspend the cells in 50 &#181;L of PBS buffer.</li>
	<li>Add equal amounts of streptavidin magnetic microbeads (1 - 2 &#181;g/106 cells) to cells in a 15-mL plastic conical tube.</li>
	<li>Gently mix well and incubate on ice for 30 min.</li>
	<li>Add 2 - 3 mL of PBS buffer to the tube.</li>
	<li>Place the tube into a magnetic stand and incubate at RT for 8 min to allow the brown microbeads to attach to the side closest to the magnet.</li>
	<li>With the tube in the magnetic stand, carefully transfer the supernatant fraction into a new sterile tube. This fraction contains the enriched CD27- na&#239;ve B cells.</li>
	<li>Add 5 mL to the tube containing the microbeads and gently resuspend the microbeads (i.e., the enriched fraction of CD27+ memory B cells)24-25.</li>
	<li>Centrifuge at 600 x g at RT for 5 min. Resuspend the pellets with RPMI 1640 medium for the downstream experiments. 
	&#8203;NOTE: Typically, ~ 30 - 60% of B cells can be purified as CD27+ memory cells from the PBMCs of a healthy donor7, 25-26.</li>
</ol>
3. Cell Sorting for the Collection of Na&#239;ve B Cells, Memory B Cells, and PBs/PCs
<ol>
	<li>Using the cells purified from step 1, determine the cell number using a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells in cold PBS buffer at the concentration of 107 per mL in a 5-mL polystyrene tube.</li>
	<li>Add 1 - 2 &#181;g of human IgG per 106 cells and incubate on ice for 10 min for the Fc block.</li>
	<li>Add 1 &#181;g each of anti-CD19-APC (clone: HIB19), anti-CD27-eFluor450 (clone: O323), and anti-CD38-PE (clone: HIT2) per 106 cells; mix well and incubate on ice for 30 min4, 27.</li>
	<li>In the last 5 min in step 3.4, add 5 &#181;L of the commercial 7-aminoactinomycin D (7-AAD).</li>
	<li>Add 2 mL of PBS to the tube, vortex, and centrifuge at 600 x g for 5 min.</li>
	<li>Resuspend the cells in sorting buffer (sterile PBS with 2% BSA and 2 mM EDTA) at a concentration of 1 - 5 x 107 cells per mL in a 15-mL tube.</li>
	<li>Filter the cells through a nylon mesh cell strainer (40 &#181;m pore size) to eliminate cell clumps.</li>
	<li>Separate the cells with a flow cytometric sorter equipped with three lasers: violet (405 nm), blue (488 nm), and red (640 nm). 
	NOTE: The blue laser alone is sufficient for 3-color flow cytometry27-28.</li>
	<li>Sort the cells into three 15-mL tubes (containing 5 mL of RPMI medium) for the simultaneous collection of na&#239;ve B cells (CD19+CD27-), memory B cells (CD19+CD27+), and PBs/PCs (CD19+CD27+/hiCD38+)3-4, 28. 
	&#8203;NOTE: Sorted na&#239;ve and memory B cells can be cultured as described in step 4.</li>
</ol>
4. In Vitro Differentiation of Isolated Human CD19+ B Cells, CD19+CD27+ Memory B Cells, and CD19+CD27- Na&#239;ve B Cells
<ol>
	<li>Using the cells purified in steps 1.19, 2.10, 2.11 and 3.10, determine the cell number with a hemocytometer or an automatic cell counter.</li>
	<li>Resuspend the cells with RPMI 1640 medium at a concentration of 1 - 10 x 105 per mL and aliquot them into the wells of a 12-well plate.</li>
	<li>Add CpG (ODN 2006) at 5 &#181;g/106 cells/mL18, 29.</li>
	<li>Culture the cells in a 37 &#176;C incubator with 5% CO2 for 5 days.</li>
	<li>Harvest the cells from each well, place them separately into 15-mL tubes, add 5 mL of PBS to each tube, and centrifuge them at 600 x g and RT for 5 min.</li>
	<li>Count the cells using a hemocytometer or an automatic cell counter. Resuspend the cells at a concentration of 1 - 10 x 105 per mL with RPMI 1640 medium.</li>
</ol>
5. ELISpot Assay
<ol>
	<li>Add 30 &#181;L of 35% ethanol in distilled water to each well of the ELISpot plates for 30 s. 
	NOTE: When pipetting, avoid touching the membrane in the wells at all times.</li>
	<li>Invert the ELISpot plates to remove the ethanol.</li>
	<li>Put 150 &#181;L of autoclaved ddH2O into each well and incubate them at RT for 5 min to flush off the residual ethanol; follow with a wash of sterile PBS at RT for 3 min. 
	NOTE: Steps 5.1 to 5.3 may be optional, depending on the manufacture of the plates.</li>
	<li>Put 50 &#181;L of 5 &#181;g/mL polyclonal F(ab&#39;)2 fragment of anti-human Ig (IgG + IgM + IgA) (in PBS) into each well of the ELISpot plates and incubate them at 4 &#176;C overnight (preferred) or 37 &#176;C for 2 h11. 
	NOTE: Seal the edges of plates with Parafilm until use.</li>
	<li>Invert the plates to remove unbound Abs, add 200 &#181;L of PBS to each well, and incubate them twice at RT for 3 min each time.</li>
	<li>Add 200 &#181;L of PBS with 5% BSA (or RPMI 1640 medium) to each well for blocking, and incubate them at RT for 2 h.</li>
	<li>Invert the plates to remove the blocking buffer. Wash each well twice with 200 &#181;L of PBS, as in step 5.5.</li>
	<li>Add 100 &#181;L of RPMI 1640 medium to each well and incubate them at 37 &#176;C.</li>
	<li>When ready to seed the cells, invert the plates to remove the RPMI 1640 medium.</li>
	<li>Seed 100 &#181;L (5 x 104), 50 &#181;L (2.5 x 104), and 25 &#181;L (1.25 x 104) of the cells (from steps 1.19, 2.10, 2.11, 3.10, and 4.6) into the wells of an ELISpot plate. With RPMI 1640 medium, bring the volume to 150 &#181;L/well. 
	NOTE: A minimum of one or two 2-fold serial dilutions for plating the cells is recommended.</li>
	<li>Incubate the plates in a 37 &#176;C incubator with 5% CO2 for 8 - 14 h18. Avoid moving the plates during incubation.</li>
	<li>Invert the plates to remove the cells and RPMI 1640 medium.</li>
	<li>Add 200 &#181;L/well of PBS-T (PBS with 0.05% Tween 20) and incubate them 5 times at RT, each time for 3 min. Invert the plate to remove the wash buffer between each of the 5 washes.</li>
	<li>Add either goat anti-human IgG-alkaline phosphatase (AP), Fc&#947;-specific Abs (for IgG detection, 1:5,000 in PBS-T) or goat anti-human IgM-AP, Fc&#181; fragment-specific Abs (for IgM detection, 1:5,000 in PBS-T) into the designated wells and incubate at RT for 2 h in the dark.</li>
	<li>Wash each well twice with 200 &#181;L of PBS, as in step 5.5.</li>
	<li>Add 50 &#181;L/well of bromochloroindolyl phosphate-nitro blue tetrazolium (BCIP/NBT) substrate solution. Purple-colored spots normally appear in 5 - 15 min.</li>
	<li>Add 100 &#181;L/well of ddH2O to prevent the over-development of spots.</li>
	<li>Rinse the plates with running tap water after the complete development of all spots.</li>
	<li>Remove the underdrain of the plates and allow them to air dry in the dark.</li>
	<li>Count the spots using an automated plate reader with an image acquisition/analysis unit (e.g., an automatic scanner or manually via a dissecting microscope). 
	NOTE: The plates can be stored at RT in the dark and analyzed later.</li>
</ol>

The author declares no competing financial interests.

Isolation and Purification of Human Peripheral Blood B Cells
Normally, RBCs can be efficiently ruptured and cleared by lysis buffer (step 1.2). It is important not to incubate PBMCs with the RBC lysis buffer longer than 5 min, as cell viability might be affected by the ammonium chloride. Alternatively, RBCs and platelets can be simultaneously removed by the following protocol.
Mix fresh whole blood with acid-citrate-dextrose (ACD) buffer (39 mM citric acid, 75 mM sodiu...

B cells play a central role in the development of humoral immunity. They initially develop in the bone marrow and enter the blood stream as na&#239;ve B cells, which can migrate into the lymphoid tissues, such as the spleen, lymph nodes, and tonsils, for further development. Upon Ag encounter, some na&#239;ve B cells migrate into lymphoid follicles, where germinal center B cells can differentiate into memory B cells and plasmablasts (PBs)/plasma cells (PCs). While most PBs/PCs egress into the blood stream, a few eventually reside in the bone marrow to undergo terminal differentiation into long-lived PCs1. B cells in circulation are heterogeneous, and at steady state, PBs/PCs are rare in peripheral blood2. As a result of the availability of lineage-specific surface markers, flow cytometry has become a popular method for the identification and characterization of the B-cell subsets in peripheral blood. An extended application of flow cytometry is the addition of a cell sorter function, which permits the separation and isolation of individual subsets of B cells with high purity. Based on the expression of specific surface receptors at different developmental stages, human circulating B cells are generally classified into three main subpopulations: na&#239;ve B cells (CD19+CD27-CD38-), memory B cells (CD19+CD27+CD38-), and PBs/PCs (CD19+CD27+CD38+)3-4 (Figure 1). Na&#239;ve B cells by nature have not encountered Ags. However, they can be differentiated into IgM+CD27+ memory B cells. Although na&#239;ve B cells are homogeneous in expressing B-cell antigen receptor (BCR)-associated molecules (e.g., CD19, CD20 and CD22) they are heterogeneous in their immunoglobulin repertoire5. The majority of CD27+ memory B cells can be differentiated into CD27+/hiCD38+ PBs/PCs6. In addition, memory B cells and PBs/PCs are polyclonal and exhibit developmental and functional heterogeneity4-7. PBs/PCs in circulation are normally short-lived and do not express CD138, but those made to settle down in the bone marrow will terminally differentiate and become long-lived. Terminally differentiated PCs express CD138 and down-regulate CD27 molecules on their surfaces8. Since both PBs and PCs are capable of secreting Abs, in many occasions they are collectively denoted as ASCs. In contrast, neither na&#239;ve B cells nor memory B cells can produce appreciable amounts of Abs9-10. Nevertheless, when isolated, both na&#239;ve and memory B cells can be differentiated into ASCs in 3 - 10 days when placed in the proper culture conditions6, 11-15. In fact, ASCs derived from in vitro differentiation share similar surface expressions of CD27 and CD38 with those directly isolated from peripheral blood6. In addition, the ASCs differentiated in vitro express a low level of surface CD20, similar that of circulating PBs/PCs6. Although the culture-derived ASCs are all short-lived, they can secrete Abs, indicating that they are functionally competent and able to contribute to the humoral immunity.
Both ELISA and ELISpot are by far the most commonly applied methods with which to obtain functional information on the humoral immune response. ELISA is a 96-well plate-based assay, and it is frequently used to measure the titers of serum Ag-specific Abs and other analytes (e.g., cytokines). It is convenient and scalable. ELISA is designed to use a solid-phase enzyme assay to detect the presence of Abs or other substances, such as serum, in a liquid sample16. The readouts from serum ELISAs have been widely used to represent the immune response of the body. A tool necessary for the acquisition of readouts from ELISA assays is a spectrophotometric microplate reader. The reader can determine the optical density (O.D.) of the end products typically resulting from the reaction of horseradish peroxidase (HRP)-conjugated detection Abs and their specific substrates17. With regard to reporting the humoral immune response, serum Ab levels determined by ELISA denote the collective, but not individual, performance of ASCs in the body. In addition, ELISA fails to take into account the participation by memory B cells, which do not secrete Abs.
Like ELISA, ELISpot is a widely used method for detecting and monitoring the immune response in peripheral blood samples17-18. ELISpot is a technique related to a sandwich ELISA. In it, cells are placed into the polyvinylidene difluoride (PVDF) membrane-backed wells of 96-well microplates for a short-term culture. The ELISpot assay is analogous to performing western blotting on a microplate and developing the spots on the PVDF membrane in each well. An automated ELISpot reader system or a stereomicroscope for manual counting is required. The main advantage of ELISpot in detecting an immune response is its superb sensitivity in the quantification of ASCs and cytokine-secreting cells. It reports their functional activities in humoral and cellular immunity, respectively. In the measurement of humoral immune function, serum Ab levels determined by ELISA and the number of ASCs enumerated by ELISpot are often correlated, but the data readouts from these two assays have some differences in functional implications19-20. The main advantage of ELISpot is its sensitivity of method. The level of serum Ab titers as reported by ELISA is presented semi-quantitatively as O.D. readouts, denoting the relative Ab level, or more quantitatively, as concentration readouts when a known amount of the proper isotypes of Abs is included for reference. In contrast, the results of ELISpot are presented as the absolute number of ASCs in a cell pool of interest (e.g., unfractionated peripheral blood mononuclear cells (PBMCs) and purified B cells from PBMCs). ELISpot can detect a single ASC, but ELISA requires Ab amounts from ASCs to reach optimized assay-dependent concentrations prior to measurement. Hence, ELISpot is obviously superior to ELISA in sensitivity of quantification. Moreover, ELISpot is also suitable for quantifying the in vitro differentiated ASCs from activated memory B cells. Memory B cells do not secrete Abs but can differentiate into ASCs upon activation; they therefore have no contribution to serum Abs detected by ELISA. Thus, ELISpot is the method of choice in the measurement of the immune response of circulating memory B cells after activation in culture. It allows for the monitoring of the maintenance of long-term humoral immunity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer K2E</td>
			<td>BD Biosciences</td>
			<td>367525</td>
			<td>10 ml tube</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>endotoxin-free</td>
		</tr>
		<tr>
			<td>Trypan blue 0.5% solution</td>
			<td>Biological Industries</td>
			<td>03-102-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>IMag Human B lymphocyte enrichment set</td>
			<td>BD Biosciences</td>
			<td>558007</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated CD27 mAb</td>
			<td>Biolegend</td>
			<td>302804</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>Streptavidin magnetic microbeads</td>
			<td>BD Biosciences</td>
			<td>9000810</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>BD Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue nylon mesh cell strainer, 40 &#956;m</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD19-APC</td>
			<td>Biolegend</td>
			<td>302212</td>
			<td>clone HIB19</td>
		</tr>
		<tr>
			<td>Anti-human CD27-eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0279-42</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>Anti-human CD38-PE-Cy7</td>
			<td>Biolegend</td>
			<td>303516</td>
			<td>clone HIT2&#160;</td>
		</tr>
		<tr>
			<td>Anti-human CD38-PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>560677</td>
			<td>clone HIT2&#160;</td>
		</tr>
		<tr>
			<td>Anti-human CD45-FITC</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td>clone HI30</td>
		</tr>
		<tr>
			<td>Anti-human CD45-FITC</td>
			<td>BD Biosciences</td>
			<td>555482</td>
			<td>clone HI30</td>
		</tr>
		<tr>
			<td>Anti-mouse/rat/human CD27-PerCP Cy5.5</td>
			<td>Biolegend</td>
			<td>124213</td>
			<td>clone LG.3A10</td>
		</tr>
		<tr>
			<td>Anti-human CD27-PerCP Cy5.5</td>
			<td>BD Biosciences</td>
			<td>65429</td>
			<td>clone L128</td>
		</tr>
		<tr>
			<td>Anti-human CD19-FITC</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-064</td>
			<td>clone LT19</td>
		</tr>
		<tr>
			<td>Anti-human CD19-FITC</td>
			<td>GeneTex</td>
			<td>GTX75599</td>
			<td>clone LT19</td>
		</tr>
		<tr>
			<td>Anti-human CD20-FITC</td>
			<td>BD Biosciences</td>
			<td>555622</td>
			<td>clone 2H7</td>
		</tr>
		<tr>
			<td>biotinylated anti-human CD27</td>
			<td>Biolegend</td>
			<td>302804</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>biotinylated anti-human CD27</td>
			<td>eBioscience</td>
			<td>13-0279-80</td>
			<td>clone O323</td>
		</tr>
		<tr>
			<td>7-aminoactinomycin D (7-AAD)</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>CpG (ODN 2006)&#160;</td>
			<td>InvivoGen</td>
			<td>tlrl-2006</td>
			<td>type B CpG</td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>PeproTech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-10</td>
			<td>PeproTech</td>
			<td>200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-21</td>
			<td>PeproTech</td>
			<td>200-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human sCD40L</td>
			<td>PeproTech</td>
			<td>310-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein A of S. aureus Cowan (SAC)</td>
			<td>Sigma-Aldrich</td>
			<td>82526</td>
			<td></td>
		</tr>
		<tr>
			<td>Pokeweed mitogen (PWM)</td>
			<td>Sigma-Aldrich</td>
			<td>L9379</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiScreen filter plates, 0.45 &#181;m pore size</td>
			<td>Merck Millipore</td>
			<td>MSIPS4510</td>
			<td>sterile, clear 96-well filter plate with hydrophobic PVDF membrane</td>
		</tr>
		<tr>
			<td>BCIP/NBT solution</td>
			<td>Sigma-Aldrich</td>
			<td>B6404</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT single reagent, alkaline phosphatase substrate</td>
			<td>Merck Millipore</td>
			<td>ES006</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG, Fc fragment</td>
			<td>Jackson ImmunoResearch</td>
			<td>009-000-008</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2 fragment of goat anti-human Ig (IgG+IgM+IgA)</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-006-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG-alkaline phosphatase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgM-alkaline phosphatase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-055-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG-peroxidase, Fc&#947; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgM-peroxidase, Fc&#181; fragment specific</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-035-095</td>
			<td></td>
		</tr>
		<tr>
			<td>BD ELISPOT AEC substrate kit</td>
			<td>BD Biosciences</td>
			<td>551951</td>
			<td></td>
		</tr>
		<tr>
			<td>C.T.L. ImmunoSpot analyzer</td>
			<td>C.T.L.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the isolation, differentiation, and quantification of human antibody-secreting b cells from blood: elispot as a functional readout of humoral immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

PBMCs were depleted of RBCs and adherent cells (steps 1.2 to 1.7). An aliquot (2 x 106) of cells were subjected to a flow cytometric analysis to illustrate the populations of na&#239;ve B cells, memory B cells, and PBs/PCs in peripheral blood (Figure 1). In this donor&#39;s PBMCs, about 10% of the lymphocytes were CD19+ B cells. In the B-cell compartment, the percentage of CD19+CD27- na&#239;ve B cells was around 50%. On the oth...

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The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

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JoVE Journal

Immunology and Infection

15.1K Views.  National Taiwan University. Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

Video: The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...