Figure 2
Figure 2. Sepsis induces increase of primitive cells with LSK phenotype. CD1 mice challenged with burn and inoculation of the PA14 (PA14) or 33C7 (33C7) strain, burn only (B), and normal controls (N) were killed at the indicated time points. BM cells were stained with antibodies directed to Lin+ markers and to Sca1 and c-Kit markers. Samples were analyzed by multicolor flow cytometric analysis. (A) Dot blots show Sca1 and c-Kit expression on gated Lin− cells in N, B, and PA14 mice at 24 hours after challenge in a representative experiment. Numbers indicate percentage of cells within the Lin− gate. (B) Line graph indicates percentage of LSK cells on overall BM with exclusion of the R1 region in Figure 1A (to avoid relative increase of percentage due to loss of the FCSlow/SSChigh population) in a representative experiment. Values are averages of 4 to 8 mice. Time 0 is the average of 8 normal animals. (C) Bar graph shows average fold increase in LSK absolute number at 24 hours from challenge. In each column values are the average of 10 mice compared with normal controls in 3 independent experiments. Error bars indicate SE. LPS: *P = .03; PA14: *P = .01. (D) Bar graph indicates percentages of LSK cells in the S+G2M phase of the cell cycle, as determined by Hoechst staining. Values indicate average of 3 mice at 24 hours from challenge in a representative experiment. Error bars indicate SD. *P < .01. (E) Long-term culture with limiting dilution was performed on BM cells to quantify the frequencies of hematopoietic progenitors and stem cells. Data indicate frequency of CAFCs and are represented as means ± SD. (F) Bar graph indicates percentage of Lin+ c-Kit+ cells in N, B, and PA14 mice at 24 hours from challenge. Values indicate average of 6 mice in a representative experiment at 24 hours from challenge. Error bars indicate SD. *P < .001. (G) Measure of LSK cell output. Bar graph shows ratio of percentages of Lin+Kit+ (Kit+) over LSK cells (r = Kit+/LSK). Percentages of each population were measured on identical gates to equal total BM excluding R1, as in Figure 1A. Values indicate average of 6 mice in 2 independent experiments at 24 hours from challenge. Error bars indicate SD. *P = .016.

Sepsis induces increase of primitive cells with LSK phenotype. CD1 mice challenged with burn and inoculation of the PA14 (PA14) or 33C7 (33C7) strain, burn only (B), and normal controls (N) were killed at the indicated time points. BM cells were stained with antibodies directed to Lin+ markers and to Sca1 and c-Kit markers. Samples were analyzed by multicolor flow cytometric analysis. (A) Dot blots show Sca1 and c-Kit expression on gated Lin− cells in N, B, and PA14 mice at 24 hours after challenge in a representative experiment. Numbers indicate percentage of cells within the Lin− gate. (B) Line graph indicates percentage of LSK cells on overall BM with exclusion of the R1 region in Figure 1A (to avoid relative increase of percentage due to loss of the FCSlow/SSChigh population) in a representative experiment. Values are averages of 4 to 8 mice. Time 0 is the average of 8 normal animals. (C) Bar graph shows average fold increase in LSK absolute number at 24 hours from challenge. In each column values are the average of 10 mice compared with normal controls in 3 independent experiments. Error bars indicate SE. LPS: *P = .03; PA14: *P = .01. (D) Bar graph indicates percentages of LSK cells in the S+G2M phase of the cell cycle, as determined by Hoechst staining. Values indicate average of 3 mice at 24 hours from challenge in a representative experiment. Error bars indicate SD. *P < .01. (E) Long-term culture with limiting dilution was performed on BM cells to quantify the frequencies of hematopoietic progenitors and stem cells. Data indicate frequency of CAFCs and are represented as means ± SD. (F) Bar graph indicates percentage of Lin+ c-Kit+ cells in N, B, and PA14 mice at 24 hours from challenge. Values indicate average of 6 mice in a representative experiment at 24 hours from challenge. Error bars indicate SD. *P < .001. (G) Measure of LSK cell output. Bar graph shows ratio of percentages of Lin+Kit+ (Kit+) over LSK cells (r = Kit+/LSK). Percentages of each population were measured on identical gates to equal total BM excluding R1, as in Figure 1A. Values indicate average of 6 mice in 2 independent experiments at 24 hours from challenge. Error bars indicate SD. *P = .016.

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