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3 protocols using cd62l pe

1

Ovalbumin-Loaded Silica Nanoparticles for Immunotherapy

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Rhodamine B isothiocyanate
(RITC), (3-aminopropyl)trimethoxysilane (APTMS), cethyltrimethylammonium
bromide (CTAB), ammonium hydroxide, tetraethylorthosilicate (TEOS),
and ovalbumin (OVA) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). HCl, methanol, and ethyl acetate were purchased from SamChun
Chemical (Seoul, Korea). Bovine serum albumin (BSA) was purchased
from Millipore (Billerica, MA, USA), CpG oligodeoxynucleotide was
purchased from Bioneer (Daejeon, Korea). Recombinant murine GM-CSF
was purchased from Peprotech (Rocky Hill, NJ, USA). Antibodies against
the following proteins were used: CD11c-APC, MHC class-II-FITC, CD86-Vioblue,
MHC class-I(H-2Kb)/SIINFEKL-PE-Vio770, CD3e-FITC, CD4-PE-Vio770,
CD8-APC, IFN-γ-FITC, CD44-Vioblue, and CD62L-PE were purchased
from Miltenyi Biotec (Bergisch Gladbach, Germany) and H-2Kb/SIINFEKL tetramer-PE was purchased from MBL life science (Woburn,
MA). IL-12 and TNF-α ELISA kits were purchased from BD science.
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2

Detailed Lymphocyte Characterization and nAb Quantification

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Heparinized blood was utilized for a 24 h whole blood peptide stimulation assay and for isolation of peripheral blood mononuclear cells using standard density gradient centrifugation protocols. Ethylenediaminetetra-acetic acid whole blood samples were stained and analyzed via flow cytometry (MACSQuant 10, Miltenyi Biotec, Bergisch Gladbach, Germany) to determine lymphocyte counts and immune cell subsets using the following conjugated antibodies: CD8-VioBlue, CD14-VioGreen, CD3-VioGreen, CD3-FITC, CD4-FITC, CD4-PE, CD62L-PE, CD20-PerCP, PD-1-PerCP, CD45RA-PerCPVio770, CD45-APC, and CD56-APCVio770 (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of all CD45+ lymphocytes expressing the surface marker of interest were multiplied by the total lymphocyte count to enumerate each lymphocyte subset per unit of whole blood. Serum was isolated from each timepoint and frozen for future analysis to determine SARS-CoV-2 nAb titers using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA).
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3

Multi-Parameter Flow Cytometry for Immune Cell Enumeration

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Whole blood samples were labeled with directly conjugated antibodies for multi-parameter flow cytometry to enumerate CD45+CD14+ monocytes and CD45+CD14- lymphocytes subsets, as previously described (20 (link)). Briefly, 100µL of EDTA whole blood was incubated with the following antibodies CD8-VioBlue, CD14-VioGreen, CD3-FITC, CD4-PE, CD62L-PE, CD20-PerCP, CD45RA-PerCPVio770, CD45-APC, and CD56-APC-Vio770 (Miltenyi Biotec Inc., Gernany) for 30 min at room temperature and then lysed (RBC lysis buffer; eBioscience, San Diego, CA) for 20 min at room temperature followed by three wash cycles. For lymphocyte and monocyte enumeration, whole blood samples were labeled with CD14 and CD45 only, underwent a lyse-no-wash procedure and adjusted for the dilution factor. The total cell numbers of each lymphocyte subset were determined by multi-parameter flow cytometry (MACSQuant 10; Miltenyi Biotec Inc. Bergisch Gladbach, Germany) by multiplying the percentage of all lymphocytes expressing the surface markers of interest by the total lymphocyte count.
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