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3 protocols using cd3e fitc

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

Characterizing Leukemic Blast Cell Populations in Mice

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Mice of generation x + 1 with signs of emerging hind limb paralysis or elevated CD19+ blast levels in the peripheral blood were anesthetized with ketamine/xylazine, and the animal was exsanguinated via the retrobulbar venous plexus. After cervical dislocation for confirmation of death, spleen and femoral bones were dissected and collected in cold PBS. Bone marrow and spleen cells were isolated as described before (Richter et al. 2020 (link)). For flow cytometric characterization of the blast population using FACSVerse (Beckton Dickinson) and FACSuite software, the following antibodies were used: CD3e-FITC, CD11b-FITC, CD8a-PE (all Miltenyi Biotec), CD34-FITC, CD45R-FITC, CD3e-PE, Sca-1-PE, CD4-APC, c-kit-APC, CD45-PerCP-Cy5.5 (all BD), CD19-PE, IgM-APC (all Biolegend). Doubling times of leukemic cell populations were calculated from peripheral blood blast frequencies using the following formula with t1 and t2 indicating the age of the animal in days at the respective time points of sample analysis: Doublingtime=t2-t1×log(2)log(blastfrequency(t2))-log(blastfrequency(t1))
Cytospins were prepared from spleen and bone marrow cell suspensions and stained as described before (Richter et al. 2019 (link)). For monitoring of CD19+ blast frequencies, blood (< 50 µl) was sampled from the tail vein, and CD19+, c-kit+ and Sca-1+ cell populations were quantified as described above.
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3

Immune Cell Profiling in Lungs

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To analyze immune lung infiltration, lung suspensions were stained as previously described [20 (link)] using the following directly conjugated antibodies: CD3e FITC (Miltenyi, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany, clone 145-2C11); CD11b PE (BD Biosciences, San Jose, CA USA, clone M1/70); CD11c PECY7 (Thermo Fisher Scientific-eBiosciences, clone N418); CD45 APCeFluor780 (Thermo Fisher Scientific-eBioscience, clone 30-F11); CD49b PE (Miltenyi, clone DX5); CD69 APC (Miltenyi, clone H1.2F3); CD103 APC (Miltenyi, clone REA789); B220 PERCPVio700 (Miltenyi, clone RA3-6B2); and mPDCA-1 PE (Miltenyi, clone JF05-1C2.4.1). A purified rat anti-mouse CD16/CD32 MAb (Thermo Fisher Scientific-eBioscience, clone 93) was used to block nonspecific binding to mouse Fc receptors. The cells were analyzed using a FACSCanto flow cytometer (BD Biosciences) and FlowJo software (TreeStar). All analyses were performed using gating on CD45+ live cells after doublet exclusion.
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