Anti mcd45 fitc clone 30f 11

Patient-derived xenograft ALL cells were thawed, washed with PBS and 1x10⁷ cells were injected in NOD/SCID (NOD.CB17-Prkdcscid/J) mice (The Jackson Laboratory, Bar Harbor, ME, USA) by the tail vein. ALL engraftment was monitored as previously described [26 (link)] and outlined below. Successfully engrafted mice were sacrificed, ALL cells were collected from spleen and liver and 1x10⁷ cells were immediately injected by the tail vein in eight secondary non-irradiated recipient mice for the subsequent experiments. Animals were monitored every 7 days for ALL engraftment as follows: blood was collected by retro-orbital bleeding into EDTA containing tube, mononuclear cells were isolated by ficoll centrifugation and the presence and quantity of ALL cells was analyzed by flow cytometry in a FACSCanto II equipment (Becton Dickinson, Franklin Lakes, NJ), using anti-hCD45-PE (clone HI30, BD Pharmingen, San Diego, CA or EXBIO, Prague, Czech Republic) and anti-mCD45-FITC (clone 30F-11, BD Pharmingen). When human CD45(+) cells reached ≥ 0.5% of peripheral blood cells in half of the animals, mice were randomly distributed into the different treatment groups (n = 4/each group). Mice were treated intraperitoneally with 10 mg/Kg of SB225002 or vehicle once a day, 5 days a week, for 4 weeks. Kaplan-Meier survival curves were compared using the Log-rank test.

After transplantation, animals were monitored every 7 days for ALL engraftment and progression. Blood was collected by retro-orbital bleeding into EDTA containing tube. Plasma was separated for ELISA and mononuclear cells were isolated by density gradient centrifugation using ficoll. ALL cells were detected by flow cytometry [7 (link)] in a FACSCanto II equipment (Becton Dickinson, Franklin Lakes, NJ), using anti-hCD45-PE (clone HI30, BD Pharmingen, San Diego, CA or EXBIO, Prague, Czech Republic) and anti-mCD45-FITC (clone 30F-11, BD Pharmingen).
For evaluation of ALL engraftment into organs, animals were sacrificed in an isoflurane chamber. Peripheral blood was immediately collected by cutting the renal vein. Bone marrow cells were obtained by flushing the femoral bones with PBS. Liver and spleen (whole organs) were mechanically homogenized and suspended with PBS. Post-ficoll mononuclear cells were analyzed as above. Total numbers of cells obtained were analyzed by flow cytometry.