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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board
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 min. 
	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 magnet.</li>
	<li>With the tube remaining in the magnetic stand, carefully transfer the supernatant into a new sterile 15-mL tube.</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. 
	NOTE: Typically, 1 - 5 x 105 B cells with a purity greater than 95% from 10 mL of peripheral blood can be isolated.</li>
</ol>

<table><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>Dye</td></tr><tr><td>IMag Human B lymphocyte enrichment set</td><td>BD Biosciences</td><td>558007</td><td>Reagent</td></tr><tr><td>Streptavidin magnetic microbeads</td><td>BD Biosciences</td><td>9000810</td><td>Reagent</td></tr><tr><td>15 ml Falcon tubes</td><td>BD Falcon</td><td>352196</td><td>Plastic ware</td></tr><tr><td>Biotinylated CD27 mAb</td><td>Biolegend</td><td>302804</td><td>Clone O323</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></tbody></table>

isolation and purification of b cells from human peripheral blood

Source: Tzeng, S. J. <a href="https://app.jove.com/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the isolation of B cells from human peripheral blood using biotinylated antibodies specific for non-B cells and streptavidin-conjugated magnetic microbeads.

Isolation and Purification of B Cells from Human Peripheral Blood

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

To perform immunomagnetic negative separation of B cells, collect human peripheral blood in an anticoagulant-containing tube to prevent blood clotting. Add red blood cell, RBC, lysis buffer to lyse the RBCs without affecting other blood cells.
Centrifuge to sediment the intact, denser blood cells from the less dense lysed RBCs, which remain in the supernatant. Discard the supernatant. Resuspend the pellet in fresh medium.
Transfer the cells to a culture plate. Incubate to allow the macrophages and monocytes to adhere to the plate while the non-adherent cells, including B cells, remain in suspension.
Collect the cells in suspension into a fresh tube. Centrifuge to pellet the cells and resuspend them in a buffer.
Add a cocktail solution containing biotinylated antibodies specific for blood cells other than B cells. The antibodies bind to antigens on the surface of non-B cells, leaving the B cells unlabeled.
Centrifuge. Discard the supernatant containing unbound antibodies.
Add a solution of streptavidin-conjugated magnetic beads. During incubation, the streptavidin on the microbeads binds tightly to the biotinylated antibodies bound to non-B cells, forming complexes.
Place the tube in a magnetic field to adhere the magnetic beads-bound non-B cells to the tube walls, leaving the B cells in suspension. Transfer the supernatant containing pure B cells to a tube.
Centrifuge and resuspend the B cells in medium for further downstream analysis.

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Isolation and Purification of B Cells from Human Peripheral Blood

Transkrypcja

Isolation and Purification of B Cells from Human Peripheral Blood