Anti cd45 antibody

Colon tissues were fixed in 4% paraformaldehyde in PBS, and the fixed sections were incubated in 3% H₂O₂ solution in PBS at room temperature for 10 min. Antigen retrieval was performed in sodium citrate buffer (0.01 M, pH 6.0) in a microwave oven at 1000 W for 3 min. Nonspecific antibody binding was blocked by incubation with 5% normal goat serum in PBS for 1 h at 25 °C. Slides were stained overnight at 4 °C with anti-claudin-3 antibody (Invitrogen, 34–1700, 1:200 dilution) and anti-CD45 antibody (BioLegend, 103102, 1:1000 dilution). The slides were subsequently washed and incubated with biotin-conjugated secondary antibodies for 30 min, and then with horseradish peroxidase streptavidin (HRP Streptavidin) for 30 min (SPlink Detection Kits; ZSGB-BIO, SP-9001 or SP-9002). The sections were developed using a 3,3’-Diaminobenzidine (DAB) substrate kit (ZSGB-BIO, ZLI-9017) and counterstained with hematoxylin. Images were captured using an Olympus BX600 microscope and a SPOT Flex camera. ImagePro Plus was used for further quantification of the DAB intensity in the image.

Flow cytometry was applied to collected CD45⁺ single cells from samples with anti-CD45 antibody (Biolegend). Data analysis was performed with FlowJo (version 10). CD45⁺ single cells were subsequently used for scRNA-seq or CyTOF.

Phagocytosis of live C. albicans cells was measured with GFP-expressing C. albicans (CaGFP) cells (47 (link)). Freshly isolated BM cells were stained with anti-CD45 antibody (BioLegend), were untreated, or were treated with 1,000 U/ml murine IFN-β, and 3 × 10⁵ BM cells were incubated with 1.5 × 10⁶ of GFP-expressing C. albicans cells. After 20 min, phagocytosis was stopped with paraformaldehyde. Samples were analyzed with a FACSCalibur flow cytometer by calculating the percentage of CD45-positive cells that were also GFP positive. A modification of the published assay to measure phagocytosis of heat-killed C. albicans was used (81 (link)). BM cells were left untreated or stimulated with murine IFN-β (1,000 U/ml). C. albicans cells were heat inactivated at 95°C for 30 min, stained with SYBR-Safe DNA stain (Thermo Fisher), and washed, and 1 × 10⁶ yeast cells were added to 1 × 10⁶ prepared BM cells. After 30 min, propidium iodide (Invitrogen) was added to quench extracellular green fluorescence. Green fluorescence in the BM cells was measured with a FACSCalibur flow cytometer (BD Biosciences).

After isolation of BM cells from donor mice, flow cytometric analysis was performed to compare the BM cell populations of the BMT and c‐BMT methods. After lysing RBCs with ammonium chloride, cells were filtered through a cell strainer (100 μm, BD Falcon). Cells (1.0 × 10⁷) were suspended in 100 μL of 2% FBS/PBS and then incubated with anti‐leptin receptor (LepR) biotinylated antibody (R&D Systems, Minneapolis, MN, USA; BAF497, 1:100), anti‐CD51 antibody (BioLegend, San Diego, CA, USA; 104105, 1:100), anti‐CD45 antibody (BioLegend, 103111, 1:100), anti‐TER‐119 antibody (BioLegend, 116211, 1:100), and anti‐CD31 antibody (BioLegend, 102509, 1:100) for 60 minutes on ice. Then cells were washed and incubated with streptavidin‐BV421 antibody (BioLegend, 405226, 1:1000) for 20 minutes on ice. For RUNX2 detection, cells were fixed and permeabilized with a transcription factor buffer set (BD Biosciences, San Jose, CA, USA) and incubated with an anti‐RUNX2 antibody (Cell Signaling, Danvers, MA, USA; #12556 S, 1:200) for 30 minutes on ice. Then cells were washed and incubated with anti‐rabbit antibody (Cell Signaling, #4412 S, 1:400) for 30 minutes on ice. Fluorescence was detected using a MACSQuant (Miltenyi Biotec, Bergisch Gladbach, Germany). Data analysis was performed using FlowJo software (BD Biosciences).

CD4 T cells were purified from lungs of convalescent mice (60 days post challenge) or mice immunized with a wP vaccine (14 days after the second dose of immunizations) or from the spleens of naïve mice or mice immunized with aP or wP vaccines (14 days after the second dose of immunization). Note: there were insufficient T cells in the lungs of naïve or aP-immunized mice to perform adoptive transfer experiments from the lungs of these mice. Convalescent mice or wP-immunized mice were injected with an anti-CD45 antibody (BioLegend) i.v. 10 min prior to euthanasia. Lung mononuclear cells were pooled from 20 wP-immunized and 20 convalescent mice and prepared as described above, followed by cell separation over continuous 40% Percoll gradient (GE Healthcare). CD45iv^- CD4 T cells were sorted using BD FACSAriaII sorter (BD Biosciences). Spleens from 10 naïve, aP- and wP-immunized mice were pressed through a 70-μm cell strainer to obtain a single-cell suspension. CD4 T cells were sorted by negative selection (Miltenyi Biotec). Naïve mice were irradiated (4.5 Gy) 1 d before cell transfer using a Gammacell irradiator. A total of 4 × 10⁵ splenic CD4 cells and 2 × 10⁵ lung CD45iv^–CD4⁺ T cells were transferred i.v. 1 d before challenge. PBS-injected mice served as controls.

We employed an established technique to distinguish tissue-resident from circulating CD4 T cells [44 (link)]. Briefly, naïve or convalescent mice or mice immunized with aP or wP vaccines were injected with an anti-CD45 antibody (BioLegend) intravenously (i.v.) 10 min prior to euthanasia. Circulating lymphocytes are exposed to the antibody and are labelled CD45iv⁺, whereas tissue-resident cells are “protected” and remain CD45iv⁻. “Tissue-resident CD4 T cells” are defined through the expression of CD4 and lack of in vivo labelling of CD45 after i.v. injection of mice with anti-CD45 10 min prior to euthanasia.