Anti human cd45 fitc clone 2d1

For flow cytometry-based evaluation of the tumor cell frequency in the bone marrow and spleen, 1 × 10⁵–1 × 10⁶ cells in 100 µL were stained with anti-human CD45-FITC (clone 2D1) and anti-human CD19-PE (clone 4G7; both Becton Dickinson, Heidelberg, Germany) at 4 °C for 15 min and washed twice (180× g, 8 min) in cold PBS before measuring using FACSCalibur or FACSVerse and CellQuest Pro or FACSuite software tools (all Beckton Dickinson), respectively. For determination of the peripheral blood blast frequencies, 20–60 µL of blood were collected from the tail vein and stained as mentioned above. Subsequently, erythrocytes were lysed during 15 min of incubation with 2 mL erythrocyte lysis buffer. Cells were washed twice in 2 mL PBS and analyzed by flow cytometry.

For patient-derived xenograft (PDX) model generation 2.5 × 10⁶ cells per primary sample were orthotopically xenografted into 8–12 week old male or female NOD scid gamma mice (NOD.Cg-Prkcd^scid Il2rg^tm1Wjl/SzJ, NSG, Charles River Laboratories, Sulzfeld, Germany). Tumor cell engraftment was regularly evaluated by peripheral blood flow cytometry (anti-human CD45-FITC (clone 2D1) and anti-human CD19-PE (clone 4G7)) measured using FACSVerse and FACSuite software, all Becton Dickinson, Heidelberg, Germany). Mice were euthanized by cervical dislocation when a blast frequency of 30% in peripheral blood was achieved and then bone marrow and spleen cells were isolated. Tumor cells were then serially transplanted into the next PDX generation until generation 3. Mice were bred and housed under specific pathogen-free conditions with access to water and standard chow ad libitum. All experiments were carried out in a laboratory setting and no intervention was performed within the animal housing and breeding rooms. Experiments were approved by the review board of the federal state Mecklenburg-Vorpommern, Germany (reference number: LALLF MV/7221.3–1.1-002/15).