Statistical analysis was performed using Prism software (La Jolla, CA, USA). The Harboe method has been established for determining hemoglobin concentrations in solution using spectrophotometric measurement at 415 nm and has been validated for assessing hemolysis in red cell
samples [14] and [15]. We adapted this method for estimating erythroid cell concentrations in unlysed culture samples and determining erythroid proliferation in a non-invasive manner. Hemoglobin shows maximum light absorption between 400 and 420 nm and we found absorbance at 405 nm and 415 nm to correlate linearly (R2 = 0.9999) allowing the use of 405 nm absorbance filters commonly available on standard plate readers ( Fig. 1a). We established that the lysis of erythrocytes is not necessary for reliable hemoglobin quantification selleck chemicals llc and that cell suspensions could be directly subjected to spectrophotometric measurement ( Fig. 1b, R2 = 0.9905). Initial assay set-up was performed using samples of native erythrocytes isolated from donor blood suspended in PBS and absorbance measurements at 405 nm were found to correlate linearly
(R2 = 0.998) with manual cell counts ( Fig. 1c). Using the function obtained from the linear fit of such an erythrocyte standard curve using GraphPad software, cultures could be expressed selleck as ‘erythrocyte equivalents’ based on their absorbance (erythrocyte equivalents/ml = (5,413,000 ± 91,210) × A405 − (154,700 ± 80,730)). Absorbance measurements were obtained from in vitro erythroid cultures at various time points of culture using a plate reader pre-heated to 37 °C, and plates were agitated to disperse cells evenly in the microwells before measurement. The absorbance values were corrected using the absorbance of the medium of each condition and normalized to a positive control culture on the same plate to determine the hemoglobinization as percentage of the positive control that in turn correlated with the cell expansion (Fig. 2). Hemoglobinization begins at the proerythroblast stage and two thirds
of a cell’s total hemoglobin are produced by the erythroblast while the remaining third is synthesized at the reticulocyte stage Methisazone [35]. In culture, cells contained detectable amounts of hemoglobin from day 8 after seeding into erythroid medium, showed strong increase in hemoglobinization over the next 7 days and reached a plateau thereafter. Absorbance measurements based on hemoglobin remained stable over extended periods of time showing only slight decreases in absorbance after further 10 days (Fig. 3), indicating that this molecule is not readily degraded even when it is released into the culture supernatant upon cell death and rupture. Cell concentrations and absorbance measurements for erythroid cultures correlated linearly and while standard deviations were larger than for native red blood cell samples, these varied comparably for both measurement principles due to higher biological variation between triplicate wells.
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