This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody's isotype, which correlates with the B cells' secretory activity.
Protocol
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. 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 review boards. In this study, the protocol to use human blood in a demonstration of the results of flow cyt…
Representative Results
Figure 1. Illustration of the Gating Strategy to Separate the Naïve B cells, Memory B cells, and PBs in the PBMCs. (A) Cells (2 x 106) were stained with 2 µg each of CD19-APC, CD27-eFluor450, and CD38-PE mAbs (step 3.4). The lymphocyte compartment was gated (G1) on the dot plot for forward scattering (FSC) versus side scattering (SSC). (B)…
Divulgaciones
The authors have nothing to disclose.
Materials
BD Vacutainer K2E
BD Biosciences
367525
10 ml tube
Ficoll-Paque Plus
GE Healthcare
17-1440-02
Endotoxin-free
Trypan blue 0.5% solution
Biological Industries
03-102-1B
IMag Human B lymphocyte enrichment set
BD Biosciences
558007
Biotinylated CD27 mAb
Biolegend
302804
Clone O323
Streptavidin magnetic microbeads
BD Biosciences
9000810
15 ml Falcon tubes
BD Falcon
352196
Blue nylon mesh cell strainer, 40 μm
BD Falcon
352340
Anti-human CD19-APC
Biolegend
302212
Clone HIB19
Anti-human CD27-eFluor 450
eBioscience
48-0279-42
Clone O323
Anti-human CD38-PE-Cy7
Biolegend
303516
Clone HIT2
Anti-human CD38-PE-Cy7
BD Biosciences
560677
Clone HIT2
Anti-human CD45-FITC
Biolegend
304006
Clone HI30
Anti-human CD45-FITC
BD Biosciences
555482
Clone HI30
Anti-mouse/rat/human CD27-PerCP Cy5.5
Biolegend
124213
Clone LG.3A10
Anti-human CD27-PerCP Cy5.5
BD Biosciences
65429
Clone L128
Anti-human CD19-FITC
Miltenyi Biotec
130-098-064
Clone LT19
Anti-human CD19-FITC
GeneTex
GTX75599
Clone LT19
Anti-human CD20-FITC
BD Biosciences
555622
Clone 2H7
Biotinylated anti-human CD27
Biolegend
302804
Clone O323
Biotinylated anti-human CD27
eBioscience
13-0279-80
Clone O323
7-aminoactinomycin D (7-AAD)
BD Biosciences
559925
CpG (ODN 2006)
InvivoGen
tlrl-2006
Type B CpG
Recombinant human IL-2
PeproTech
200-02
Recombinant human IL-10
PeproTech
200-10
Recombinant human IL-21
PeproTech
200-21
Recombinant human sCD40L
PeproTech
310-02
Protein A of S. aureus Cowan (SAC)
Sigma-Aldrich
82526
Pokeweed mitogen (PWM)
Sigma-Aldrich
L9379
MultiScreen filter plates, 0.45 µm pore size
Merck Millipore
MSIPS4510
Sterile, clear 96-well filter plate with hydrophobic PVDF membrane
BCIP/NBT solution
Sigma-Aldrich
B6404
BCIP/NBT single reagent, alkaline phosphatase substrate
Merck Millipore
ES006
Human IgG
Jackson ImmunoResearch
009-000-003
Human IgG, Fc fragment
Jackson ImmunoResearch
009-000-008
F(ab')2 fragment of goat anti-human Ig (IgG+IgM+IgA)
Jackson ImmunoResearch
109-006-127
Goat anti-human IgG-alkaline phosphatase, Fcγ fragment specific
Jackson ImmunoResearch
109-055-008
Goat anti-human IgM-alkaline phosphatase, Fcµ fragment specific
Jackson ImmunoResearch
109-055-095
Goat anti-human IgG-peroxidase, Fcγ fragment specific
Jackson ImmunoResearch
109-035-008
Goat anti-human IgM-peroxidase, Fcµ fragment specific