Figure 1.
Determination of the optimal DMSO concentration for cryopreservation of bone marrow cells.
Bone marrow (BM) cells were resuspended in heat-inactivated fetal bovine serum containing 0, 5, 10, 15 or 20% DMSO and then stored in liquid nitrogen for one week. Cells were thawed and counted to estimate the number of BM cells after thawing (white bars) and were then cultivated in bone marrow differentiation media. After 7 days of differentiation, the cultures were washed with ice cold PBS to remove undifferentiated cells and the bone marrow derived macrophages (BMDM) were detached from the plates and counted (black bars). The dotted line represents the initial number of frozen BM cells per aliquot.
Figure 2.
Fresh or cryopreserved bone marrow cells show similar expression of cell markers.
Cells from fresh or cryopreserved (frozen) bone marrow were harvested and labeled with phycoerythrin (PE)-conjugated Abs against CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, Sca-1 and PDGF or control IgGs and analyzed by FACS. (A) Dot plot showing the two gates analyzed (G1 and G2). (B and C) Averages and standard deviation of the triplicates show the percentages of the cells positive for each marker within the gated population: G1 (B) or G2 (C). Data are one representative experiment out of two independent experiments performed. No statistically significant differences were detected between fresh or frozen BM cells (P>0.05).
Figure 3.
Numbers of frozen bone marrow cells per differentiated bone marrow-derived macrophages.
Bone marrow cells (BM) were obtained from a single mouse and frozen at concentrations of 1.4×106, 2.7×106, 4.2×106, 6.4×106 or 1.4×107 cells/vial. The cells were thawed and cultivated in bone marrow differentiation media for 7 days. The cultures were washed with ice cold PBS to remove undifferentiated cells and the bone marrow derived macrophages (BMDM) were detached from the plates and counted. The figure shows the non-linear correlation between the number of frozen BM cells (x-axis) and the number of differentiated BMDM (y-axis).
Figure 4.
Frozen bone marrow cells maintain differentiation competence over time.
Bone marrow cells were frozen at a concentration of 4×106 cells per vial at different times and maintained in liquid nitrogen. Eight aliquots were simultaneously thawed and the cells were cultivated in bone marrow differentiation media. After 7 days of differentiation, the cultures were washed with ice cold PBS to remove undifferentiated cells and the bone marrow derived macrophages (BMDM) were detached from the plates and counted.
Figure 5.
Bone marrow-derived macrophage obtained from fresh or cryopreserved bone marrow cells similarly express CD80 and CD86 in response to LPS and Legionella pneumophila.
BMDMs obtained from fresh or cryopreserved (frozen) bone marrow cells were left untreated (NS), stimulated with 1 µg/ml LPS or infected with L. pneumophila at an MOI of 0.5, 1 and 2. Cultures were cultivated in non-tissue culture-treated 6 well-plates at a density of 1×106 BMDM per well and stimulated/infected for 18 hours. Cells were stained for CD11b, F4/80, CD80 and CD86 and evaluated by flow cytometry. Plots were gated on CD11b+ cells. Shown are the percentage of cells F4/80+CD80+ (A) and F4/80+CD86+ (C). The mean fluorescence intensities are shown for CD80 (B) and CD86 (D). Data are representative of those found in three independent experiments. Asterisks indicate statistically significant differences in relation to NS (P<0.05). No statistically significant differences were detected between BMDM obtained from fresh or frozen BM cells (P>0.05).
Figure 6.
Bone marrow-derived dendritic cells obtained from fresh or cryopreserved bone marrow cells showed similar expression of CD40, CD86 and MHC-II in response to LPS.
Bone marrow-derived dendritic cells (BMDCs) obtained from fresh or cryopreserved (frozen) bone marrow cells were either left untreated or stimulated with 1 µg/ml LPS for 24 h. BMDCs were stained for CD11b, CD11c, CD40, CD86 and MHC-II and analyzed by flow cytometry. Plots were gated on CD11b+ cells. Shown are the percentages (A) and mean fluorescence intensities (B) of CD11c+ cells expressing CD40, CD86 or MHC-II in response to LPS treatment. Data are representative of those found in two independent experiments. No statistically significant differences were detected between BMDCs obtained from fresh or frozen BM cells (P>0.05).
Figure 7.
Bone marrow-derived macrophages obtained from fresh or frozen bone marrow cells respond similarly to infection with Legionella pneumophila.
(A) Bone marrow-derived macrophages (BMDM) obtained from fresh (open squares) or cryopreserved (frozen; closed circles) bone marrow (BM) cells were infected with wild type L. pneumophila (Lp01, continuous lines) or with flagellin-deficient L. pneumophila (flaA-, dashed lines). BMDM were cultivated and infected in 24-well plates with the indicated Legionella strain at an MOI of 0.015. After 48 hours post-infection, cells were lysed and the colony-forming units (CFU) were estimated for each of the triplicate wells. (B) Fresh (white bars) or frozen (black bars) BMDMs were infected with Lp01 at an MOI of 10 for 1 hour. Cultures were vigorously washed with PBS and further incubated for a total of 9 hours of infection. Bacteria were stained with a rabbit anti-L. pneumophila polyclonal antibody and the number of bacteria per individual LCV were enumerated using an epifluorescence microscope. Shown are the frequencies of LCV containing: 1; 2–3; 4–10; and more than 10 bacteria (>10). At least 100 LCV in independent cells were scored per each of the triplicate coverslips. Data are representative of those found in three (A) or two (B) independent experiments. No statistically significant differences were detected between BMDM obtained from fresh or frozen BM cells (P>0.05).
Figure 8.
Bone marrow-derived macrophage obtained from fresh or cryopreserved bone marrow cells respond similarly to the infection with Leishmania amazonensis.
Bone marrow-derived macrophages (BMDM) obtained from fresh or cryopreserved (frozen) bone marrow cells were infected with L. amazonensis promastigotes constitutively expressing the GFP at an MOI of 10 parasites per cell. After 48 h and 96 h of infection, the percentages of L. amazonensis-GFP+ and propidium iodide-positive cells were estimated by flow cytometry. (A) Percentage of GFP+ BMDM obtained from fresh (white bars) and frozen (black bars) BM cells. (B) Mean fluorescence intensities (MIF) for GFP signal. (C) Representative images of cultures infected for 48 h e 96 h with L. amazonensis GFP. (D) FACS profiles of BMDM obtained from fresh and frozen BM cells infected with L. amazonensis GFP. Shown are the percentages of cells with signal for GFP and PI. (E) Bar graphics indicating the percentage of propidium iodide-positive cells in uninfected cultures (control), cells treated with 0.05% Tween 20 (Tween) or after infection with L. amazonensis for 48 and 96 hs. Data are representative of those found in three independent experiments. No statistically significant differences were detected between BMDM obtained from fresh or frozen BM cells (P>0.05).