Recipient NOD/SCID mice received a single dose of 3.5Gy of total body irradiation (TBI, 137Cs source) followed 24 hours later with an i.v. injection of 1x105 bone marrow cells isolated either from the femurs of Ames dwarf (df/df) or littermate control (+/df) mice. Peripheral blood was collected from the tail vein into heparinized microcapillary tubes (Fisher Scientific; Pittsburg, PA, USA) 1 and 2 months post transplantation. Following lysis of red blood cells using lysis buffer (0.155M NH4Cl, 0.01M KHCO3, 0.1mM EDTA in H2O; Sigma-Aldrich), cells were washed, blocked with both human and mouse gamma globulin, stained with PE-conjugated anti-mouse H-2Kk, and fixed prior to flow analysis on a FACSCalibur utilizing BD CellQuest™ Pro software to determine percent H-2Kk+ cells. This was done to determine the percentage of cells that were Ames in origin since Ames mice are H-2Kk+ and NOD/SCID mice are not. Three female littermate controls (+/df) and dwarf (df/df) mice were used for these experiments. The bone marrow from each mouse was transplanted into 5 recipients. Therefore, there was a total of 15 NOD/SCID mice each that received either +/df bone marrow or df/df bone marrow.
Heparinized microcapillary tubes
Manufactured by Thermo Fisher Scientific
Sourced in United States
Heparinized microcapillary tubes are small, cylindrical glass or plastic tubes used to collect and transport small volumes of blood samples. The tubes are coated with heparin, an anticoagulant, to prevent the blood from clotting during collection and transportation.
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Lab products found in correlation
2 protocols using heparinized microcapillary tubes
1
Ames Dwarf Bone Marrow Transplantation in NOD/SCID Mice
2
Preparation of Hemoglobin Derivatives for MR Analysis
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This study received ethics approval from the local Institutional Review Board of the National Healthcare Group. K2 EDTA-anticoagulated whole blood samples were washed and re-suspended with phosphate buffer saline (PBS). Informed consent was obtained from all subjects involved in this study. All blood samples were either used immediately or kept at 4 °C and used within three to four days (unless mentioned otherwise) of collection before the micro MR analysis. To induce the Hb into various derivative states, the blood samples were incubated with the desired chemical as mentioned in the Text (e.g., sodium nitrite) and finally washed to remove the chemical residual. Heparinized micro capillary tubes (Fisher Scientific, PA) were used to transfer the processed blood and finally spun down at 6000 × g for 1 min to obtain packed red blood cells for MR measurements.
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