23 gauge needle

At day 21 and 42, two birds per replicate (n = 16) were randomly selected and weighed. Before killing, 5 mL of blood was collected from the brachial vein using serum collection tubes (23-gauge needle; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA), for analysis of serum metabolite (albumin, total protein, glucose, cholesterol, uric acid, creatinine), liver enzymes (Alkaline Phosphatase (ALP), Alanine Aminotransferase (SGPT or ALT), Aspartate Aminotransferase (SGOT or AST) and Lactate Dehydrogenase (LDH)) and to perform immunological assessment (NDV and IBD titers) according to the procedure recommended by commercial analytical kits manufacturer (Sigma-Aldrich, Saint Louis, MO, USA). Immediately after the collection of blood, serum was separated by gravimetry (30 min on ice) and samples were centrifuged (3000× g at 4 °C for 15 min), aliquoted (1.2 mL) in 1.5 mL centrifuge tubes (Fisherbrand, Fisher Scientific, Hampton, NH, USA) and stored at −24 °C in a freezer (Thermo Fisher Scientific, Waltham, MA, USA) for further analysis. The immune responsiveness was assessed in terms of humoral immune responses, mainly for antibody production to NDV and IBD vaccines. The antibody titer was calculated as per the equations provided by the manufacturer.

For each anaerobic liquid sample, 50 μl of overnight (16-h) precultures were diluted in 5 ml of potassium phosphate (100 mM)-buffered (pH 7.4) LB (7 (link), 43 (link)) in a Balch tube and then incubated at 37°C with shaking at 250 rpm for 2.5 h to early to mid-exponential phase (OD₅₀₀, ∼0.5). Balch tubes containing subcultures were then transferred into the anaerobic chamber (80% N₂, 15% CO₂, and 5% H₂) and sealed with rubber stoppers to maintain anoxia. Anaerobic tubes were incubated with shaking at 250 rpm at 37°C throughout the period of the survival assay. To measure CFU from the anaerobic culture, 10 to 50 μl of culture was aspirated from the tube with a 1-ml syringe (Care Touch) fitted with a 23-gauge needle (Becton, Dickinson), and then serially diluted in LB down to 10⁻⁶ CFU and plated on 1% tryptone plates for CFU counting.

Bone marrow cells were flushed from tibias/femurs of mice using a 23-gauge needle (Becton Dickinson). LDMNCs were isolated by density gradient using Histopaque-1119 (Sigma), centrifuging for 30 minutes (1800 rpm, no brake); LDMNCs were removed from the interface. Murine cytospins were made by resuspending LDMNCs in PBS and centrifuging onto slides (450rpm, 5 minutes) on a Shandon Cytospin-3 Cytocentrifuge (Thermo). For competitive transplant studies, 0.5-1 million donor test LDMNCs from wt or Fancc-/-; Mad2+/- (C57Bl/6J, CD45.2+) mice were transplanted with wt donor competitor LDMNCs (BoyJ, CD45.1+) via tail-vein injection into eight-week old female B6.SJL-Ptprc^aPepc^b/BoyJ mice that underwent whole-body split-dose 1100 rads irradiation (700 rads/400 rads, 4 hours apart). A competitive transplant design was used to ensure recipient survival to evaluate engraftment and propagation of malignancy post-radiation.