Methocult gf m3434 methylcellulose medium

The CFC assay was described previously [25] (link). Briefly, freshly isolated total bone marrow cells were suspended in Methocult GF M3434 methylcellulose medium (StemCell Technologies, Vancouver, B.C., Canada). Triplicate cultures were set up for each specimen according to the manufacturer's instructions. After 14 days of culture, the total colony- forming unit (CFU) were counted.
The CAFC assay was described previously [25] (link). Briefly, a monolayer of FBMD-1 stromal cells was plated in 96-well plates and grown to confluent; whole bone marrow cells were seeded at 81,000, 27,000, 9000, 3000, 1000 or 333 cells per well in CAFC medium (Iscove's MDM supplemented with 20% horse serum, 10⁻⁵ M hydrocortisone, 10⁻⁵ M 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin). Twenty replicate wells per cell number were counted. The frequency of CAFC was determined on days 7, 14, 21, 28 and 35. Wells were scored positive if at least one phase dark hematopoietic clone (containing five or more cells) was seen. The frequencies of CAFCs were calculated using the L-Calc program (StemCell Technologies).

CFC assays were performed using BMMNCs in MethoCult GF M3434 methylcellulose medium (Stem Cell Technologies, Canada). Colony-forming unit granulocyte-macrophage (CFU-GM), colony-forming unit erythroid (CFU-E), burst-forming unit erythroid (BFU-E), and colony-forming unit mix (CFU-Mix) were counted on days 5, 7, 9, and 12, respectively, using a microscope according to the manufacturer's protocol.

CS (molecular weight [Mw]: ≈30 × 10⁴ Da, 92% deacetylated) was obtained from Shenzhen Zhongfayuan Biological Technology Co. Ltd. (Shenzhen, China). SCS and SCOS were synthesized according to previous study.^[57] The mass average molecular weight (M_w) of SCS and SCOS was measured using Viscotek 270max (Malvern), and the M_w was 12 073 and 1979 Daltons, respectively. The Fourier transform infrared spectroscopy of CS, COS, SCS, and SCOS was measured using Nicolet 6700 (Thermo Scientific). Sulfo‐Cy5‐labeled SCS and SCOS were synthesized according to the manufacturer's instructions. Sulfo‐Cy5 (HY‐D0819A) was purchased from Med Chem Express Co. Ltd. (Shanghai, China). Gelatin sponge was purchased from Xiang'en Medical Technology Development Co. Ltd. (Jiangxi, China). Recombinant human bone morphogenetic protein‐2 (RhBMP‐2; E. coli derived) was provided by Shanghai Rebone Biomaterials Co., Ltd. (Shanghai, China). MethoCult GF M3434 methylcellulose medium was obtained from Stem Cell Technologies (Vancouver, Canada). Red blood cells lysis solution was obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Cell staining buffer was obtained from Biolegend Biotechnology Co., Ltd. (San Diego, CA, USA).

CFU assays were performed by culturing BM-MNCs in MethoCult GF M3434 methylcellulose medium (Stem Cell Technologies, Vancouver, BC) according to the manufacturer's protocol. Colonies of colony-forming unit-granulocyte macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) were scored on day-7, while colonies of CFU-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were enumerated on day-12 after incubation. CAFC assays were conducted to evaluate HSC activity in vitro as we reported previously [14 (link)]. And day-14 and day-35 CAFC frequencies were determined to measure the functions of HPCs and HSCs, respectively, as described previously [13 (link)–15 (link)].

BM-derived LSKs were obtained from middle-aged female or male mice per flow cytometry cell sorting as described above. In order to assay the potential effects of FSH and testosterone, the original growth factor concentrations in the MethoCult GF M3434 methylcellulose medium (STEMCELL Technologies) were halved by mixing with the MethoCult M3234 medium at 1:1 ratio. LSK cells (5000 cells/well) were then resuspended in 1.5 ml of the growth factor-reduced culture medium containing mouse recombinant FSH (R and D Systems, 0.2 or 1 μg/ml) or testosterone (Sigma-Aldrich, 0.2 or 1 μg/ml). The length of the in vitro clonogenic assay with FSH or testosterone was optimized in separated preliminary experiments. Total cells were harvested after 9 days of culture, and colony forming and differentiation were evaluated under a microscope and then analyzed using flow cytometry after staining with anti-mouse antibodies against CD45 (for all hematopoietic cells), Gr-1 (for myeloid cells), and B220 (for B cells). Each experiment was performed multiple times in duplicates.