Methocult h4434 classic

CD34⁺ cells (1.5 × 10³ cells), treated or not with EVs for 20 h, were plated in two 35-mm dishes in MethoCult Classic H4434 (Stem Cell Technologies, Vancouver, BC, Canada) and incubated at 37°C in a humidified atmosphere at 5% CO₂. MethoCult classic medium H4434 already contains SCF, IL3, GM-CSF, EPO, and FBS. After another 14 days, colonies were counted with an inverted microscope (Zeiss, Germany). The absolute number of colonies, as a sum of the two dishes, was calculated. The percentage of specific colonies was calculated on the total number of colonies for each experimental condition.

In separate experiments without calcein, cells were treated with ultrasound ± microbubbles and samples were harvested for both viability assays and studies on intracellular signaling. Cell concentration was determined by manual counting using a haemocytometer. This was done immediately after treatment with ultrasound and after 24 h of culture. Apoptotic cell death was assessed by adding 0.01 mg/mL Hoechst 33342 (Merck KGaA, Darmstadt, Germany) to cells after 24 h of culturing. Cells were fixed in 4% formaldehyde and imaged by fluorescence microscopy. The total number of cells was determined in Fiji [20 (link)] by adjusting the image brightness and contrast followed by a manual threshold then using the “Analyze particles” function. Apoptotic cells were counted manually using the “Cell counter” plugin in Fiji. Colony forming assay (Methocult™ Classic H4434; Stemcell™ Technologies, Vancouver, BC, Canada) was used to evaluate the proliferation of the MOLM-13 cells. Cells were seeded immediately after treatment and colonies were counted after 7–10 days incubation. All experiments were performed in triplicate.