The virulent L. major isolate MHOM/IL/81/FE/BNI (Solbach et al., 1986 (link); Stenger et al., 1996 (link)) was maintained through serial passage in BALB/c mice in vivo and cultured in vitro in Novy-Nicolle-MacNeal blood agar slants in RPMI1640 medium supplemented with 10% new born calf serum, penicillin/streptomycin, non-essential amino acids and 10 mM HEPES, all supplied by Invitrogen Life Technologies (Mount Waverly, Australia). For infection, stationary phase L. major promastigotes were used between passage 2 and 6 and 3 × 106 parasites were injected in a volume of 40 μl into one hind footpad. The infection site was monitored daily and the increase in lesion size was determined weekly by measuring the footpad thickness with a metric caliper (Kroeplin Schnelltaster, Schluechtern, Germany). The percentage of increase in footpad thickness was determined by the formula (thickness of infected footpad minus thickness of non-infected footpad/mean thickness of non-infected footpad) × 100. The parasite burden (per gram of tissue) was determined at day 28 post infection (p. i.) using a limiting dilution method and L-Calc software version 1.1 (Stem Cell Technologies, www.Stemcell.com ) which performs a generalized Pearson Chi-squared test (Wilhelm et al., 2001 (link)).
L calc software version 1
Manufactured by STEMCELL
L-Calc software version 1.1 is a cell counting and analysis tool. It provides automated cell counting capabilities.
Automatically generated - may contain errors
3 protocols using l calc software version 1
1
Virulent L. major Infection in BALB/c Mice
2
Clonogenic Outgrowth Assay and Insertional Mutagenesis
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Outgrowth was determined initially by microscope, followed by MTT assay. Prior research had demonstrated that 4× SD of all readings reproduced the microscopic scoring and was determined as the threshold value distinguished from background absorbance readings in the MTT assay. If the measured absorbance was above the threshold, then the well was scored as positive in the MTT matrix. Clonal outgrowth was independently screened by two technologists in four categories. Category 1 is a robust outgrowth with a visible pH change of the medium. Category 2 is obvious outgrowth of cells. Category 3 shows some level of proliferation and may be assigned to category 2 in case of a positive result in the MTT assay. Category 4 shows no cell growth and scores as negative in the MTT assay.
The RF was calculated from the number of wells above threshold using the L-Calc software (version 1.1) from STEMCELL Technologies according to Poisson distribution. The risk of insertional mutagenesis was correlated to the number of vector copies per cell, and RFs were normalized to the VCN.
The RF was calculated from the number of wells above threshold using the L-Calc software (version 1.1) from STEMCELL Technologies according to Poisson distribution. The risk of insertional mutagenesis was correlated to the number of vector copies per cell, and RFs were normalized to the VCN.
3
Leukemia-Initiating Cell Frequency Quantification
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Pairwise comparisons were performed using the Mann-Whitney U test, unless otherwise specified. The Kaplan-Meier method with log-rank test was used to compare differences between survival curves. Spearman correlation was used for tests of relationships. Leukemia-initiating cell (LIC) frequencies were calculated with L-Calc™ Software Version 1.1 (STEMCELL Technologies). Unless otherwise indicated, data are expressed as mean ± standard error of the mean (s.e.m.). A P-value of less than 0.05 was defined as statistically significant; statistical significance is shown as *P<0.05, **P<0.01 and ***P<0.001.
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