Pe conjugated anti cd117 c kit

All BM cells were flushed out from the femur by normal saline and the spleen and lung was ground into single cells and filtered by nylon net (106 μm). For analysis of MKs in peripheral blood, 50 µL of blood was taken from the ophthalmic venous plexus and added to an EP tube prefilled with sodium citrate for mixing. After being washed with PBS twice, the cell density was adjusted to 1 × 10⁶ per sample by counting on a hematology analyzer. Then, according to the purpose of the experiment, the samples were labeled with the corresponding antibodies for 30 min in the dark, respectively. It is worth noting that if PI is required for relabeling, 300 µL PI stain solution is incubated for another 15 min after completing other antibody labeling. Finally, marker expression was analyzed using BD FACSCanto II flow cytometry (BD Biosciences, San Jose, CA, USA). Specific flow antibody information were as follows: FITC conjugated anti-CD41 (BioLegend, San Diego, CA, USA), PE‑conjugated anti‑CD117 (c‑Kit, BioLegend, San Diego, CA, USA), PE‑conjugated anti‑CD61 (BD Biosciences, San Jose, CA, USA), APC conjugated anti-CD62p (BioLegend, San Diego, CA, USA), FITC conjugated anti-CD42d (BioLegend,y,San Diego, CA, USA).

Cells were preincubated 10 min with anti-CD16/32 FcγII/III (BD Bioscience) to prevent nonspecific binding, washed and stained for surface markers, fixed in Fix/Perm buffer (BD Bioscience), and permeabilized in permeabilization buffer (BD Bioscience) for 1 h with antibodies. For intracellular staining, GolgiStop (BD Bioscience) was added 6 h before Fc block, fixation, and permeabilization (BD Bioscience). Primary antibodies for flow cytometry were: PerCP/Cy5.5-conjugated anti-CD45 (BD Biosciences), APC-conjugated anti-CD11b (BD Bioscience), PE-conjugated anti-CD117/c-Kit (BioLegend), FITC-conjugated anti-FcεRIα (BioLegend), PE/Cy7-conjugated anti-T1/ST2 (IL-33R; eBioscience), APC-conjugated anti-IL-22 (eBioscience), and PE/Cy7-conjugated anti-IL-13 (eBioscience). Acquisition was with a BD FACSCanto instrument. Data analyses used FlowJo software (Tree Star).

For the analysis of MKs in BM, spleen and blood cells, total BM cells were washed out of the femur with saline solution, and the spleen was ground into single cells and filtered by nylon net. Red blood cells (RBCs) were removed from the cell samples with RBC lysis buffer (Beijing 4 A Biotech, Beijing, China). For analysis of MKs in peripheral blood, 50 μL of blood was taken from the ophthalmic venous plexus and added to an EP tube prefilled with sodium citrate for mixing. The cell density was adjusted to 100×10⁴ per sample by counting on a hematology analyzer. The samples were labeled with FITC-conjugated anti-CD41 (BioLegend, San Diego, CA, USA), PE-conjugated anti-CD117 (c-Kit, BioLegend, San Diego, CA, USA) and FITC-conjugated anti-CD41 (BioLegend, San Diego, CA, USA) and PE-conjugated anti-61 (BD Biosciences, San Jose, CA), FITC-conjugated anti-CD41 and PE-conjugated anti-CD62P (BioLegend, San Diego, CA, USA) on ice for 30 min in the dark. Flow cytometry was performed using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA).