Cd235a fitc

In the differentiation experiment, LSCs were cultured with Iscove’s Modified Dulbecco’s Medium (IMDM) medium (Gibco, Carlsbad, CA, USA) containing 10% FBS, 100 ng/mL recombinant human stem cell factor (rhSCF), 100 ng/mL rhFlt3 and 100 ng/mL for recombinant human thrombopoietin (rhTPO) with 5% CO₂ at 37° C for 24 h. After culture, LSCs were treated with 1 μg/mL AML cell-derived EVs for 7 days. Next, LSCs were collected and labeled with CD19-PE (555413, BD Biosciences, San Jose, CA, USA), CD33-APC (561817, BD Biosciences, San Jose, CA, USA), CD3-APC (561811, BD Biosciences, San Jose, CA, USA), and CD235a-FITC (559943, BD Biosciences, San Jose, CA, USA) for 30 min. Cells were then detected using a FACS Calibur (BD Biosciences, San Jose, CA, USA) and analyzed with the Cell Quest software (BD Biosciences, San Jose, CA, USA) [33 (link)].

The fourth-generation canine ADMSCs were inoculated into 96-well plates at 5 × 10² cells per well for a total of 44 wells. Contents were taken from four wells every day, and the MTT Cell Proliferation Assay Kit (ThermoFisher Scientific, Waltham, MA, United States) was used to determine the proliferation of cells with continuous determination for 11 days.
The fourth-generation canine ADMSCs were adjusted to 2 × 10⁶ cells/mL suspension; 100 μL cell suspension was transferred into a flow cytometry tube and 5 μL CD13-FITC, CD29-FITC, CD31-FITC, CD44-FITC, CD45-PE, CD73-FITC, CD90-PE, CD105-PE, and CD235a-FITC fluorescent antibodies (BD Biosciences, San Jose, CA, United States) were added. Cells were incubated without light for 15 min and flow cytometry (CytoFLEX, Beckman Coulter, :Brea, CA, United States) was used for detection.
Dog Adipose-derived Stem Cell Adipogenic/Osteogenic/Chondrogenic Differentiation Basal Medium (CAXMD-90031¹ /CAXMD-90021² /CAXMD-90041,³ Cyagen, China) were used to induce canine ADMSCs into adipocytes/osteocytes/chondrocytes. The differentiated cells were stained with Oil Red O, Alizarin Red, and Alcian Blue, respectively.

NK cells and iRBCs were resuspended in experimental media (no human serum). 6 × 10⁵ NK cells and 2 × 10⁵ iRBCs were mixed at a 3:1 ratio in 96-well plates and incubated for 6 hr at 37°C in the absence or presence of antibodies. For experiments using plasma or serum, the total amount of plasma or serum in each condition was (20 μl plasma/serum into 200 μl media) to control for the level of plasma. 20 μl of US serum the negative control, then increasing volume of Mali immune plasma was added in (Example: 2 μl Mali plasma with 18 μl US serum totaling 20 μl plasma/serum). After a 6 hr coincubation of iRBCs and NK cells, soluble Abs were removed by a wash. This washing step removed any antibody that would bind to merozoites. A 100-fold excess of uRBCs (2 × 10⁷) relative to iRBCs was then added and cultures were maintained for an additional 16 hr at 37°C in standard parasite growth conditions. At the end of incubation, CD45- PE (BD Biosciences), CD235a-FITC, and Hoechst were used to stain NK cells, uRBCs and iRBCs (Figure 2—figure supplement 1C). Samples with NK cells but in the absence of antibodies were used as control to calculate growth inhibition.