3. Growth of pneumococcal strains is variable. Depending on
how many cells are required in downstream applications, multiple flasks per strain may be required.
4. We find that pneumococcal growth is better with the larger
surface area provided by flasks. Vented caps allow for better
aeration of culture. Satisfactory growth of cultures was
obtained without shaking of flasks.
5. It is important that antibodies are titrated for number of
PBMCs stained, especially if there are differences in PBMC
numbers between control and experiment samples. Significant
gating issues may arise including inability to visualize certain
populations if the right antibody amounts are not used.
6. We tested four different bacteria-to-PBMC ratios—1:1, 4:1,
16:1, and 64:1. No significant difference was observed
between the 16:1 and 64:1 ratios; both, however, yielded
isolation of significantly higher pathogen-specific plasmablasts
than the 1:1 and 4:1 ratios, as determined by FACS.
7. When analyzing more than one sample, a mastermix of the
antibody cocktail can be prepared.
Acknowledgments
This study was funded, in part, by research grants from John and
Michelle Bresnahan via MeningitisNow (awarded to P.R.L), an
Imperial College Confidence-in-Concept grant (Number
PS3075_WMNP awarded to P.R.L and F.A.B), a UK Biotechnology and Biological Sciences Research Council PhD studentship
(BB/R505742/1 awarded to C.A.G), and a UK Medical Research
Council Career Development Award (MR/S007490/1 awarded to
F.A.B).
References
1. Fink K (2012) Origin and function of circulating
plasmablasts during acute viral infections. Front
Immunol 3:78
2. Smith K, Garman L, Wrammert J et al (2009)
Rapid generation of fully human monoclonal
antibodies specific to a vaccinating antigen. Nat
Protoc 4(3):372
3. Lin Z, Chiang NY, Chai N et al (2014) In vivo
antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to
facilitate the isolation of rare monoclonal antibodies from human B cells. Nat Protoc 9
(7):1563
4. Mazutis L, Gilbert J, Ung WL et al (2013)
Single-cell analysis and sorting using dropletbased microfluidics. Nat Protoc 8(5):870
5. Pinder CL, Kratochvil S, Cizmeci D et al (2017)
Isolation and characterization of antigen-specific
plasmablasts using a novel flow cytometry–based
Ig
capture
assay.
J
Immunol
199
(12):4180–4188
6. Ho IY, Bunker JJ, Erickson SA et al (2016)
Refined protocol for generating monoclonal
antibodies from single human and murine B
cells. J Immunol Methods 438:67–70
18
Sara Siris et al.
how many cells are required in downstream applications, multiple flasks per strain may be required.
4. We find that pneumococcal growth is better with the larger
surface area provided by flasks. Vented caps allow for better
aeration of culture. Satisfactory growth of cultures was
obtained without shaking of flasks.
5. It is important that antibodies are titrated for number of
PBMCs stained, especially if there are differences in PBMC
numbers between control and experiment samples. Significant
gating issues may arise including inability to visualize certain
populations if the right antibody amounts are not used.
6. We tested four different bacteria-to-PBMC ratios—1:1, 4:1,
16:1, and 64:1. No significant difference was observed
between the 16:1 and 64:1 ratios; both, however, yielded
isolation of significantly higher pathogen-specific plasmablasts
than the 1:1 and 4:1 ratios, as determined by FACS.
7. When analyzing more than one sample, a mastermix of the
antibody cocktail can be prepared.
Acknowledgments
This study was funded, in part, by research grants from John and
Michelle Bresnahan via MeningitisNow (awarded to P.R.L), an
Imperial College Confidence-in-Concept grant (Number
PS3075_WMNP awarded to P.R.L and F.A.B), a UK Biotechnology and Biological Sciences Research Council PhD studentship
(BB/R505742/1 awarded to C.A.G), and a UK Medical Research
Council Career Development Award (MR/S007490/1 awarded to
F.A.B).
References
1. Fink K (2012) Origin and function of circulating
plasmablasts during acute viral infections. Front
Immunol 3:78
2. Smith K, Garman L, Wrammert J et al (2009)
Rapid generation of fully human monoclonal
antibodies specific to a vaccinating antigen. Nat
Protoc 4(3):372
3. Lin Z, Chiang NY, Chai N et al (2014) In vivo
antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to
facilitate the isolation of rare monoclonal antibodies from human B cells. Nat Protoc 9
(7):1563
4. Mazutis L, Gilbert J, Ung WL et al (2013)
Single-cell analysis and sorting using dropletbased microfluidics. Nat Protoc 8(5):870
5. Pinder CL, Kratochvil S, Cizmeci D et al (2017)
Isolation and characterization of antigen-specific
plasmablasts using a novel flow cytometry–based
Ig
capture
assay.
J
Immunol
199
(12):4180–4188
6. Ho IY, Bunker JJ, Erickson SA et al (2016)
Refined protocol for generating monoclonal
antibodies from single human and murine B
cells. J Immunol Methods 438:67–70
18
Sara Siris et al.