Drabkin s solution

Matrigel plug assay was conducted to measure the angiogenesis as previously described [29 ]. Nude mice were purchased from the National Laboratory Animal Center (Beijing, China). Four-week-old BALB/c athymic nude mice were fed with autoclaved food and water during the experiment. All the experiments with nude mice were performed strictly in accordance with the protocol approved by the Administrative Panel on Laboratory Animal Care of Shengjing Hospital. In brief, GECs re-suspended in 400 mL of solution containing 80% Matrigel at a density of 3 × 10 [5 ] were subcutaneously injected. After 4 days, plugs were removed, weighed, photographed, and collected by dissolving in 400 μm PBS (overnight incubation at 4 °C). The content of hemoglobin (Sigma) was determined by Drabkin’s solution (Sigma) according to manufacturer’s directions.

All in vivo studies were performed at the animal facility of the Universidad de los Andes (Santiago, Chile) and received approval by the ‘Universidad de Los Andes ethical committee for animal experimentation.’ In order to evaluate the angiogenic potential of MenSCs, matrigel plug assay was performed in eight-week-old NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories, Bar Harbor, ME, USA). For the matrigel plug assay, mice were randomly divided into four groups (n = 6/8 plugs per group): DMEM (as negative control), HUVEC, BM-MSCs and MenSCs. Growth factor-reduced matrigel (BD Bioscience) was mixed with (D)MEM alone (supplemented with 50 ng/ml VEGF) or 3 × 10⁶ cells (resuspended in DMEM supplemented with 50 ng/ml VEGF), and subcutaneously injected into both flanks of mice. Fourteen days later, matrigel plugs were harvested and processed for hemoglobin quantification. In brief, the matrigel plugs were homogenized in 250 μl Brij-35 solution (Sigma), cleared by centrifugation at 5,000 g for five minutes and the supernatants were collected to measure hemoglobin content with Drabkin’s solution (Sigma) according to the manufacturer’s instruction. Student’s t-test was used to calculate statistical difference. A P value of P ≤0.05 was considered to be statistically significant.

The Matrigel plug assay was performed to evaluate the angiogenic response in vivo. Matrigel Growth Factor Reduced (0.45 mL, Corning) mixed with heparin (10 U/mL) and PBS or BafA-PD (500 ng/mL) were subcutaneously injected into the flanks of 8-week-old C57BL/6J mice. On day 10 post injection, mice were sacrificed, Matrigel plugs were removed, and blood vessel formation was evaluated by quantification of hemoglobin. The hemoglobin assay was performed as follows: Matrigel plugs were soaked in an equal volume of RBC lysis buffer (Sigma-Aldrich) and incubated overnight on ice. Drabkin’s solution (Sigma-Aldrich) containing 0.3% Brij-35 was added to the samples and incubated for 30 min at room temperature. The hemoglobin concentration was determined by comparing the spectral absorption at 540 nm of the sample with that of hemoglobin standards (Sigma-Aldrich).

The samples were covered with PBS resulting in a surface-to-PBS ratio of 3 cm²/mL in screw-cap polypropylene tubes. Subsequently, pooled blood was added at a ratio of 1:7 and incubated at 37°C for 3 hours. During this time, the tubes were inverted carefully twice every 30 minutes. A sample of 1.8 ml was removed from the test tubes and centrifuged at 700–800 g for 15 minutes. Drabkin’s solution (Sigma, Germany) was added to the samples at equal volumes and incubated at room temperature for 15 minutes. Two aliquots of each sample were transferred to a microtiter plate and the absorbance was measured at 540 nm. All measurements were done in triplicates and catheters (ARROWg + ard Blue, Arrow international) and high density polyethylene films (RM-C, Hatano Research Institute) were used as hemolytic and non-hemolytic controls, respectively.

Hemoglobin levels were monitored daily by diluting 2 μL tail-vein blood in 500 μL Drabkin's solution (Sigma Aldrich, Canada). Following 15 min of incubation in the dark, 200 μL of blood was transferred into 96-well plates (Costar, USA) in duplicate and absorbance was measured at 540 nm in a microplate reader. Values were converted to g/dL using a standard curve of rat hemoglobin (Sigma Aldrich, Canada) prepared in Drabkin's solution. All samples were assessed in duplicate. To estimate the percentages of reticulocytes in blood, 1 μL of tail-vein blood was collected in 1 mL of PBS. The cell suspensions were then stained with anti-CD71-FITC antibody (BioLegend, USA) and incubated at 4°C for 30 min. Data were acquired with an Accuri C6 (Becton Dickinson, USA) and analyzed with the FlowJo software (Tree Star). In parallel, parasitemia was measured daily in methanol fixed blood smears stained with a 10% Giemsa solution in PBS during 15 minutes.