Methocult gf

Sorted cells were plated in triplicate in methylcellulose (MethoCult GF; STEMCELL Technologies) with 1% PS and incubated at 37°C, 5% CO₂ for 12 d. Hematopoietic colony types were distinguished by morphology and counted with an inverted microscope.
For all transplantations, 9-Gy irradiated (split dose) C57BL/6 female recipients were used. 2 × 10⁵ C57BL/6 spleen cells were coinjected with the sorted cell samples. Chimerism in hematopoietic tissues was assessed by semi-qPCR for the GFP transgene (eGFP FW 5′-AAACGGCCACAAGTTCAGCG-3′ and RV 5′-GGCGGATCTTGAAGTTCACC-3′), normalized to myogenin (Myo FW 5′-TTACGTCCATCGTGGACAGC-3′ and RV 5′-TGGGCTGGGTGTTTAGTCTTA-3′). Control mixes of Ly6aGFP and WT DNA were used to make a standard curve, and the trend line formula was used to calculate the percentage of reconstitution of each sample. Peripheral blood cell donor chimerism was assayed at 1 and 4 mo after injection, and mice were sacrificed for analysis of donor chimerism in all hematopoietic tissues. Recipients considered reconstituted are ≥10% donor chimerism positive. All experiments have been conducted according to Dutch law and have been approved by the animal experiments committee (Stichting DEC consult, Dier Experimenten commissie, protocol numbers 138-11-01 and 138-12-13).

BM cells (2 × 10⁴) and splenocytes (1 × 10⁶) from WT and knockout mice were seeded in semisolid Methocult GF M3434 medium containing rmSCF, rmIL-3, recombinant human (rh)IL-6, and rhEpo for detection of colony-forming units-granulocyte, monocyte and burst-forming units-erythroid (Stem Cell Technologies, 03434). Colony numbers were counted on day 7, and images for colony sizes were obtained on day 8.

Colony-forming unit-granulocyte (CFU-G), CFU-macrophage (CFU-M), CFU-granulocyte macrophage (CFU-GM) and CFU-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) were assessed by culturing sorted HSPCs from BM or spleen or 7-AAD⁺Lin⁻CD45⁺ cells from kidneys in MethoCult GF (StemCell Technologies; M3434) in duplicates. The enumeration and identification of colonies was performed after 7–9 days. Normalisation was performed to colony numbers/10³ HSPCs.

For methylcellulose assays, the cells were resuspended in StemPro-34 SFM medium at a concentration of 3 × 10⁴/mL. Five hundred microliters of cell suspension was mixed with 2.5 mL MethoCult GF+ (H4435, Stem Cell Technologies), and 1 mL was plated in duplicate 35-mm dishes. Colonies were scored after 14 days and averaged between the duplicate dishes.

To determine the growth characteristics of renin-lineage cells, bone marrow cells from Ren1^dcre/+;mT/mG reporter mice were grown in methylcellulose with medium permissive for either B-lymphocyte or myeloid-erythroid colony growth (Methocult GF medium for mouse Pre-B cells, M3630, and Methocult GF for mouse haematopoietic progenitors, M3434, StemCell Technologies Vancouver, BC, Canada). Cells were diluted in Iscove’s MDM with 5% FBS, and plated in duplicate samples at 1 × 10⁵ cells per 30 mm plate. After incubation for 14 days at 37 °C in 5% humidified CO₂, plates were visualized using inverted microscopy (Leica DMIRE2) and colony-forming units (CFUs) were scored according to standard criteria45 (link). The percent of GFP⁺ (renin-lineage) colonies was quantitated in both B-cell and myeloid/erythroid permissive culture conditions.