Single-cell sorting of plasmablasts

PBMCs were freshly isolated from blood samples collected 7 days after PfSPZ immunization and stained for viability with Aqua Live/Dead dye(Invitrogen) followed by surface-staining for the following markers: CD20-Cy7APC (BD Bioscience), CD19-FITC (BD Bioscience), CD3-Cy7PE (BD Bioscience), CD38-PE (BD Bioscience), and CD27-APC (ThermoFisher). Plasmablasts were gated as live, CD3-CD20-CD19+CD27+CD38+ and were sorted as single cells into 96-well PCR plates containing 20 μl/well of RT reaction buffer that included 5 μl of 5× First strand cDNA buffer, 0.5 μl of RNAseOut (Invitrogen), 1.25 μl of DTT, 0.0625 μl of Igepal and 13.25 μl of dH2O (Invitrogen) as previously described^{46 (link)}.

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Kisalu N.K., Idris A.H., Weidle C., Flores-Garcia Y., Flynn B.J., Sack B.K., Murphy S., Schön A., Freire E., Francica J.R., Miller A.B., Gregory J., March S., Liao H.X., Haynes B.F., Wiehe K., Trama A.M., Saunders K.O., Gladden M.A., Monroe A., Bonsignori M., Kanekiyo M., Wheatley A.K., McDermott A.B., Farney S.K., Chuang G.Y., Zhang B., Natasha K.C., Chakravarty S., Kwong P.D., Sinnis P., Bhatia S.N., Kappe S.H., Sim B.K., Hoffman S.L., Zavala F., Pancera M, & Seder R.A. (2018). A human monoclonal antibody prevents malaria infection and defines a new site of vulnerability on Plasmodium falciparum circumsporozoite protein. Nature medicine, 24(4), 408-416.

Publication 2018

Aqua Live/Dead dye CD20-Cy7APC CD19-FITC CD3-Cy7PE CD38-PE CD27-APC CD3-CD20-CD19 RNAseOut Igepal

Blood Buffer Cdna Cells Fitc Immunization

Corresponding Organization :

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fred Hutch Cancer Center, Johns Hopkins University, Center for Infectious Disease Research, Massachusetts Institute of Technology, Broad Institute

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Protocol cited in 2 other protocols

Variable analysis

independent variables

PfSPZ immunization

dependent variables

Plasmablasts (live, CD3-CD20-CD19+CD27+CD38+)

control variables

Freshly isolated PBMCs from blood samples
Staining for viability with Aqua Live/Dead dye
Surface-staining for CD20, CD19, CD3, CD38, and CD27 markers

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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