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Research ArticleMethods/New Tools, Novel Tools and Methods

High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons

Sara S. Parker, Aubin Moutal, Song Cai, Sambamurthy Chandrasekaran, Mackenzie R. Roman, Anita A. Koshy, Rajesh Khanna, Konrad E. Zinsmaier and Ghassan Mouneimne
eNeuro 13 September 2018, 5 (5) ENEURO.0135-18.2018; DOI: https://doi.org/10.1523/ENEURO.0135-18.2018
Sara S. Parker
1Department of Neuroscience, College of Science, University of Arizona, Tucson, AZ 85724
8Department of Cellular and Molecular Medicine, College of Medicine, University of Arizona, Tucson, AZ 85724
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Aubin Moutal
2Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724
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Song Cai
2Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724
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Sambamurthy Chandrasekaran
3Bio5 Institute, University of Arizona, Tucson, AZ 85724
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Mackenzie R. Roman
4Department of Molecular and Cellular Biology, College of Science, University of Arizona, Tucson, AZ 85724
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Anita A. Koshy
5Departments of Neurology, Cellular and Molecular Medicine, and Immunobiology, College of Medicine, University of Arizona, Tucson, AZ 85724
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Rajesh Khanna
6Departments of Neuroscience, Pharmacology, and Anesthesiology, Colleges of Science and Medicine, University of Arizona, Tucson, AZ 85724
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Konrad E. Zinsmaier
7Departments of Neuroscience and Molecular and Cellular Biology, College of Science, University of Arizona, Tucson, AZ 85724
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Ghassan Mouneimne
8Department of Cellular and Molecular Medicine, College of Medicine, University of Arizona, Tucson, AZ 85724
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  • Figure 1.
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    Figure 1.

    Survey of cryopreservation media for the storage of primary cortical neurons. A, Experimental design and timeline for validation of cryostored neuron performance. Neurons were concurrently plated from a fresh dissection or cryostored aliquot and evaluated on indicated DIV. B, Percentage of recovered and lost cells after cryostorage in test media CS10, CS5, SAF, or 50% FBS-40% DMEM-10% DMSO (50:40:10). Percentage of “recovered” cells was calculated with ((# cells alive + # cells dead) ÷ (# cells in initial aliquot)) × 100. Percentage of “lost” cells was calculated with 100% recovered. Mean ± SD, N = 3 source dissections. C, Post-thaw viability of cells cryostored in test media compared to the viability of freshly dissected cells as evaluated by Trypan blue exclusion. Mean ± SD, N = 3 source dissections. D, Table displaying summarized data from A, B (white columns) and calculated data (gray columns). Viable cell yield for each experiment was calculated by (percentage recovered × percentage viable) ÷ 100. Efficiency was determined by normalizing viable cell yield to the average viability of a fresh dissection. E, Immunofluorescence labeling of neuron-specific β-III-tubulin (orange) and fluorescently-conjugated phalloidin labeling of the actin cytoskeleton (blue) of DIV7 cells cryostored in test media. Scale bar = 100 μm.

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    Figure 2.

    Long-term survival of neurons cryostored in CS10 only slightly lower than a fresh dissection. A, Cell survival after cryostorage and recovery in test media compared to freshly dissected cells at DIV3. Live cells were labeled by calcein (green), and dead cells are indicated by ethidium homodimer labeling (magenta). Scale bar = 100 μm. B, Quantification of live/dead assay at DIV3. Table displays average change in percentage survival of cryostored cells versus fresh cells. Mean ± SD, N = 3 source dissections, n > 2300 cells per condition. C, D, Immunofluorescence labeling of fresh or cryostored cells fixed at DIV4. Cells were labeled with antibodies against the neuron-specific marker β-III-tubulin (C, D; orange), astrocyte marker S100 (C; blue), or microglia marker Iba1 (D; magenta). Scale bar = 100 μm. E, Percentage of cell population at DIV4 labeled with β-III-tubulin or S100. Mean ± SD, N = 3 source dissections, n > 1250 cells per condition. F, Percentage of cell population at DIV4 labeled with β-III-tubulin or Iba1. Mean ± SD, N = 3 source dissections, n > 250 cells per condition. All statistical comparisons made by unpaired parametric t test (B, E) or Mann–Whitney U test (F). n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

  • Figure 3.
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    Figure 3.

    Expression of key neurodevelopmental genes is unchanged following recovery from cryostorage. RT-qPCR of RNA samples from fresh or CS10-cryostored cortical cultures collected at DIV12. CTs were normalized to housekeeping genes and relative copy numbers were generated using 2-ΔCT × 106. Floating bar graph spans the minimum and maximum data points, vertical line denotes mean. N = 3 source dissections. All statistical comparisons made by unpaired parametric t test with Holm–Sidak correction for multiple comparisons. Multiplicity adjusted p value for each comparison listed in table (right).

  • Figure 4.
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    Figure 4.

    Cryostorage does not alter neuron morphogenesis. A, B, Immunofluorescence labeling of freshly dissected or CS10-cryostored cells, using antibodies against the dendrite marker MAP2 (magenta) and axon marker Tau (green). Cells were fixed on DIV4 (A) or DIV7 (B). Cultures grown to DIV7 were also infected with a sub-saturating titer of cytosolic TurboRFP-encoding lentivirus to aid in tracing (inverted white). Scale bar = 100 μm. C–E, Quantification of number of primary neurites (C), primary neurite length (D), and axon length (E) of traced cortical neurons at indicated timepoints and conditions. Box-and-whisker plots show data pooled from three independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (○◻△) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. N = 3 source dissections, n > 90 neurons per condition and time point. Statistical comparisons between biological replicate means made by unpaired parametric t test. F–H, Quantification of number of primary neurites (F), primary neurite length (G), and axon length (H) of freshly dissected cortical neurons compared to aliquots from the same source dissection cryostored for indicated durations, and traced at DIV7. Box-and-whisker plots show data from each experiment. n > 14 neurons per condition and time point. Inset graph plots mean for each dataset, symbols (○◻△) denote each source dissection, dashed line connects fresh-CS10 matched pairs. Statistical comparisons between matched means made by paired parametric t test. n.s. = not significant. Fresh DIV7 morphometric data for each dissection were extracted from C–E.

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    Figure 5.

    Neuron arborization and branch complexity are unaffected by cryostorage. A, Diagram of Sholl analysis with indicated metrics: enclosing radius, maximum number of intersections (Nm), critical radius (rc), and Sholl regression coefficient (k). B, C, Representative masks of neuron tracings illustrate number of intersections (spectrum scale bar) detected by Sholl analysis of DIV4 (B) and DIV7 (C) cortical neurons from indicated conditions. Scale bar = 100 μm. D, E, Sholl profiles of freshly dissected (orange) or CS10-cryostored (blue) neurons at DIV4 (D) and DIV7 (E). Data are represented as mean (solid line, left y-axis) ± SD (shading); number of neurons (n; dashed line, right y-axis). F–I, Quantification of enclosing radius (F), Nm (G), rc (H), and k (I) of Sholl-analyzed tracings at indicated timepoints and conditions. Box-and-whisker plots show data pooled from three independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (○◻△) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. N = 3 source dissections, n > 90 neurons per condition and time point. Statistical comparison between mean Sholl profiles at each radius made by unpaired parametric t test with Holm–Sidak correction for multiple comparisons (D, E), and statistical comparison between biological replicate means for Sholl metrics made by unpaired parametric t test (F–I). n.s. = not significant.

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    Figure 6.

    Synaptogenesis occurs normally in neurons following cryostorage and recovery. A–C, Neurons expressing Lifeact-mRuby2 (inverted white) from indicated conditions were fixed at DIV12 (A), DIV14 (B), and DIV16 (C), and immunolabeled using antibodies against excitatory postsynaptic marker PSD95 (NeuroMab; green), inhibitory postsynaptic marker gephyrin (GPHN; cyan), and presynaptic marker synaptophysin (SYP; magenta). Bottom panel is magnified segment of boxed dendrite region. Scale bar = 100 μm (top) and 5 μm (bottom panel). D, Density of all actin-rich protrusions from secondary dendrites at indicated timepoints and conditions. E, Ratio of dendritic spines to dendritic filopodia as determined by presence or absence of PSD95 on actin-rich protrusions at indicated timepoints and conditions. Box-and-whisker plots (D, E) show data pooled from three independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (○◻△) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. F, Percentage of PSD95-positive excitatory synapses and GPHN-positive inhibitory synapses at DIV16 from indicated conditions. Mean ± SD, N = 3 source dissections, n = 6–7 neurons per condition. All statistical comparisons between biological replicate means made by unpaired parametric t test. n.s. = not significant.

  • Figure 7.
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    Figure 7.

    Cryopreserved cortical neurons have normal spontaneous and stimulated activity. A, Resting membrane potential of fresh and CS10-cryostored neurons recorded by whole-cell patch clamp. Scatterplot of data points pooled from one to two biological replicates. Mean ± SD, N = 1–2 source dissections, n = 10–14 neurons per condition. B, Examples of whole-cell patch clamp recordings of neurons exhibiting spontaneous activity from fresh or CS10-cryostored neurons at DIV12–DIV14. C, Representative tracings of evoked APs after 100-pA current injection for 50 ms (red). D–F, Quantification of evoked AP parameters including amplitude (D), rise time (E), and half-width (F). Scatterplot of data points pooled from one to two biological replicates. Mean ± SD, N = 1–2 source dissections, n = 7–11 neurons per condition. G, Fluorescent images of calcium imaging experiments showing the representative response of CS10-cryostored neurons to the indicated trigger at DIV14. Baseline (left column) Fura-2 fluorescence F340/F380 ratio, and peak (right column) Fura-2 fluorescence F340/F380 ratio, in response to 15-s stimulation with the indicated trigger. Scale bar = 100 μm. H, Traces of mean change in Fura-2 fluorescence F340/F380 ratio over time for the indicated triggers and conditions. I, Summary bar graphs showing the peak fluorescence response (as variation from baseline, adjusted for background) for the indicated triggers. Mean ± SEM, N = 1 source dissection, n > 75 neurons per condition. J, Glutamate release by DIV14 neurons from indicated conditions in non-depolarizing solution (basal), after 15 min of stimulation with KCl (stimulated), and after return to non-depolarizing solution (post-stim). Glutamate release at each time point is presented as a percentage of the total glutamate released. K, Fold change of total glutamate released by CS10-cryostored neurons relative to fresh. Mean ± SEM, N = 3 source dissections. Statistical comparisons made by unpaired parametric t test (A, I–K) or Mann–Whitney U test (D–F). n.s. = not significant.

  • Figure 8.
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    Figure 8.

    Duration of cryostorage does not impact several essential neuronal properties but may affect electrophysiological performance. A, Viability of cells cryostored in CS10 media and recovered at various times after freezing. Dashed gray line = linear regression; yellow shaded area = 95% confidence band. N = 12 source dissections, n = 38 cryostored aliquots. Statistical analysis performed by Pearson’s correlation test. n.s. = not significant. B, RT-qPCR of RNA samples from DIV12 cortical cultures cryostored for three months or one year. CTs were normalized to housekeeping genes and relative copy numbers were generated using 2-ΔCT × 106. Floating bar graph spans the minimum and maximum data points, vertical line denotes mean. N = 2 source dissections, n = 3 thawed aliquots. Statistical comparisons made by unpaired parametric t test with Holm–Sidak correction for multiple comparisons. Multiplicity adjusted p value for each comparison listed in table (right). C–E, Quantification of number of primary neurites (C), primary neurite length (D), and axon length (E) of traced cortical neurons at indicated timepoints. Box-and-whisker plots show data pooled from three independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (○◻△) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. N = 2 source dissections, n > 65 neurons per condition and time point. Statistical comparisons between biological replicate means made by unpaired parametric t test (C–E). F, Examples of whole-cell patch clamp recordings of spontaneous activity from neurons recovered after one-year cryostorage in CS10 at DIV12–DIV14. G, Representative tracing of evoked APs after 100-pA current injection for 50 ms (red). H, Resting membrane potential of neurons recovered after one-year cryostorage in CS10 recorded by whole-cell patch clamp, alongside freshly dissected and three month-cryostored neurons reproduced from Figure 7 for comparison (separated by dashed line). Scatterplot of data points pooled from one to two biological replicates. Mean ± SD, N = 1–2 source dissections, n = 8–14 neurons per condition. I–K, Quantification of evoked AP parameters including amplitude (I), rise time (J), and half-width (K) of neurons recovered after one-year cryostorage in CS10, alongside freshly dissected and three month-cryostored neurons reproduced from Figure 7 for comparison (separated by dashed line). Scatterplot of data points pooled from one to two biological replicates. Mean ± SD, N = 1–2 source dissections, n = 7–11 neurons per condition. Statistical comparisons made by one-way ANOVA with Tukey’s test for multiple comparisons (H) or Kruskal–Wallis test with Dunn’s test for multiple comparisons (I–K). n.s. = not significant, *p < 0.05, **p < 0.01.

  • Figure 9.
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    Figure 9.

    CS10 is effective for the cryostorage of primary hippocampal neurons. A, Percentage of recovered and lost hippocampal cells after cryostorage in CS10. Percentage of “recovered” cells was calculated with ((# cells alive + # cells dead) ÷ (# cells in initial aliquot)) × 100. Percentage of “lost” cells was calculated with 100% recovered. Mean ± SD, N = 7 source dissections. B, Post-thaw viability of CS10-cryostored hippocampal cells compared to the viability of freshly dissected cells as evaluated by Trypan blue exclusion. Mean ± SD, N = 7 source dissections. C, RT-qPCR of RNA samples from fresh or CS10-cryostored hippocampal cultures collected at DIV12. CTs were normalized to housekeeping genes and relative copy numbers were generated using 2-ΔCT × 106. Floating bar graph spans the minimum and maximum data points, vertical line denotes mean. N = 3 source dissections. Statistical comparisons made by unpaired parametric t test with Holm–Sidak correction for multiple comparisons. Multiplicity adjusted p value for each comparison listed in table (right). D, Immunofluorescence labeling of freshly dissected or CS10-cryostored hippocampal neurons at DIV4, using antibodies against the dendrite marker MAP2 (magenta) and axon marker Tau (green). Scale bar = 100 μm. E–G, Quantification of number of primary neurites (E), primary neurite length (F), and axon length (G) of freshly dissected or CS10-cryostored hippocampal neurons at DIV4. Box-and-whisker plots show data pooled from four independent experiments; whiskers indicate 10th to 90th percentile, box indicates 1st to 3rd quartile, center line is median, gray points are data outside 10th to 90th percentile. Symbols (○◻△◇) denote the mean value calculated for each biological replicate, horizontal line is the grand mean. N = 4 source dissections, n > 115 neurons per condition. H, Immunofluorescence labeling of freshly dissected or CS10-cryostored hippocampal neurons expressing Lifeact-mRuby2 (inverted white) at DIV16, using antibodies against excitatory postsynaptic marker PSD95 (Novus; green), inhibitory postsynaptic marker gephyrin (GPHN; cyan), and presynaptic marker synaptophysin (SYP; magenta). Scale bar = 5 μm. I, Density of actin-rich protrusions from secondary dendrites ay DIV16 from indicated conditions. J, Percentage of PSD95-positive excitatory synapses and GPHN-positive inhibitory synapses at DIV16 from indicated conditions. Mean ± SD, N = 4 source dissections, n > 16 neurons per condition. All statistical comparisons between biological replicate means made by unpaired parametric t test (E–G, I, J). n.s. = not significant.

Tables

  • Figures
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    Table 1.

    Primary antibody source and usage

    Target nameSourceRRIDDilution
    Iba1Rb polyclonal, Wako (019-19741)RRID:AB_8395041:500
    S100Rb polyclonal, Dako (Z0311)RRID:AB_100133831:400
    Olig2Ms monoclonal, MilliporeSigma (MABN50, 211F1.1)RRID:AB_108074101:500
    β-III-tubulinRb monoclonal, Cell Signaling (5568, D71G9)RRID:AB_106945051:200
    β-III-tubulinMs monoclonal, MilliporeSigma (MAB1637, TU-20)RRID:AB_22105241:200
    TauMs monoclonal, MilliporeSigma (MAB3420, PC1C6)RRID:AB_948551:750
    MAP2Rb monoclonal, Cell Signaling (8707, D5G1)RRID:AB_27226601:200
    PSD951Ms monoclonal, UC Davis/NIH NeuroMab Facility (75-028 K28/43)RRID:AB_22929091:100
    PSD952Ms monoclonal, Novus Biologicals (NB300-556, 6G6-1C9)RRID: AB_20923661:500
    SynaptophysinCk polyclonal, Synaptic Systems (101 006)RRID:AB_27226611:500
    GephyrinRb polyclonal, Alomone (AIP-005)RRID:AB_27226621:500
    • Rb: rabbit, Ms: mouse, Ck: chicken.

    • ↵1 used in Figure 6.

    • ↵2 used in Figure 9.

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    Table 2.

    RT-qPCR primer sequences

    Target nameSequenceDirectionSource
    Eef1a1 CAACATCGTCGTAATCGGACAFwdPrimerBank, Harvard
      GTCTAAGACCCAGGCGTACTTRev 
    Rpl29 CAAGTCCAAGAACCACACCACFwdPrimerBank, Harvard
      GCAAAGCGCATGTTCCTCAGRev 
    Pax6 TACCAGTGTCTACCAGCCAATFwdPrimerBank, Harvard
      TGCACGAGTATGAGGAGGTCTRev 
    Emx1 GAAGAATCACTACGTGGTGGGFwdTerrigno et al. (2008)
      CCGTTTGTATTTTGTCCTCCGARev 
    Emx2 GGCTAGAGCACGCTTTTGAGFwdTerrigno et al. (2008)
      CACCGGTTAATGTGGTGTGTRev 
    Mtap2 CTCCTCGCAGGGGTGTATCAFwdPrimerBank, Harvard
      GTCCGTCGTGCTGAAGAGARev 
    Tbr1 CGCCCTCCTCCATCAAATCCATCGFwdTerrigno et al. (2008)
      GCAGTTCTTCTCGCAGTCCCGCRev 
    Reln TTACTCGCACCTTGCTGAAATFwdPrimerBank, Harvard
      CAGTTGCTGGTAGGAGTCAAAGRev 
    Sat2b GCCGTGGGAGGTTTGATGATTFwdPrimerBank, Harvard
      ACCAAGACGAACTCAGCGTGRev 
    Cux1 TGACCTGAGCGGTCCTTACAFwdPrimerBank, Harvard
      TGGGGCCATGCCATTTACATCRev 
    Lhx9 TCCAAAACGCACGAGCCAAFwdTerrigno et al. (2008)
      CAGGTCTGTTAAAGTGGTCGCRev 
    Lmo3 ACACGAAGGCTAACCTTATCCTFwdTerrigno et al. (2008)
      AGTTTCCCGTTACACCAAACAGRev 
    Gap43 TGGTGTCAAGCCGGAAGATAAFwdPrimerBank, Harvard
      GCTGGTGCATCACCCTTCTRev 
    Slc17a6 CTGAGAAGAAGGCTCCGCTATFwdPrimerBank, Harvard
      ATGCCGAAGGATATGCAGAAGRev 
    Slc32a1 ACCTCCGTGTCCAACAAGTCFwdPrimerBank, Harvard
      TCAAAGTCGAGATCGTCGCAGRev 
    Tubb3 GCCAAGTTCTGGGAGGTCATFwdPrimerBank, Harvard
      GGGCACATACTTGTGAGAGGARev 
    Gfap ACCAGCTTACGGCCAACAGFwdPrimerBank, Harvard
      CCAGCGATTCAACCTTTCTCTRev 
    Bcl11b CCCGACCCTGATCTACTCACFwdPrimerBank, Harvard
      GGAGGTGGACTGCTCTTGTRev 
    Nes CCCCTTGCCTAATACCCTTGAFwdPrimerBank, Harvard
      GCCTCAGACATAGGTGGGATGRev 
    Foxg1 CACTTTGAGTTACAACGGGACCFwdPrimerBank, Harvard
      CGAGTTTTGAGTCAACACGGARev 
    • View popup
    Table 3.

    Statistics table

    FigureData structureTestCI of difference of mean/medianp value  
    a2B, Fresh v CS10NormalUnpaired parametric two-tailed t test-0.2311 to 0.11680.4131  
    b2B, Fresh v CS5NormalUnpaired parametric two-tailed t test-0.4476 to -0.16190.0041  
    c2B, Fresh v SAFNormalUnpaired parametric two-tailed t test-0.6306 to -0.39990.0002  
    d2B, Fresh v 50:40:10NormalUnpaired parametric two-tailed t test-0.5666 to -0.34370.0003  
    e2E, Fresh v CS10NormalUnpaired parametric two-tailed t test4.188 to 22.270.0153  
    f2E, Fresh v CS5NormalUnpaired parametric two-tailed t test4.207 to 46.590.0292  
    g2E, Fresh v SAFNormalUnpaired parametric two-tailed t test34.23 to 78.220.0021  
    h2E, Fresh v 50:40:10NormalUnpaired parametric two-tailed t test19.57 to 78.160.0098U 
    i2F, Fresh v CS10NongaussianMann–Whitney U test-2.87 to 0.3846 (median)0.40002 
    j2F, Fresh v CS5NongaussianMann–Whitney U test-2.22 to 0.3846 (median)0.99994 
    k2F, Fresh v SAFNongaussianMann–Whitney U test-3.76 to 0.3846 (median)0.99994 
    l2F, Fresh v 50:40:10NongaussianMann–Whitney U test-1.11 to 0.3846 (median)0.40002 
          nadj p value
    m3NormalUnpaired parametric t test, multiple comparisons using Holm–SidakN/A0.0050-
    0.8389
    18 genes0.0857
    -0.9999
    n4C, DIV4NormalUnpaired parametric two-tailed t test-1.982 to 2.5650.7397  
    o4C, DIV7NormalUnpaired parametric two-tailed t test-1.337 to 3.2330.3135  
    p4D, DIV4NormalUnpaired parametric two-tailed t test-12.02 to 4.7850.2980  
    q4D, DIV7NormalUnpaired parametric two-tailed t test-17.28 to 11.790.6278  
    r4E, DIV4NormalUnpaired parametric two-tailed t test-29 to 78.070.2601  
    s4E, DIV7NormalUnpaired parametric two-tailed t test-119.5 to 133.50.8870  
    t4FNormalPaired parametric two-tailed t test-2.378 to 0.86450.1824  
    u4GNormalPaired parametric two-tailed t test-15.4 to 6.6310.2288  
    v4HNormalPaired parametric two-tailed t test-121.6 to 1290.9097  
          nadj p value
    w5DNormalUnpaired parametric t test, multiple comparisons using Holm–SidakN/A0.0018-
    0.9973
    1207 radii0.8864
    -0.9999
    x5ENormalUnpaired parametric t test, multiple comparisons using Holm–SidakN/A0.0128-
    0.9974
    1610 radii>0.9999
    y5F, DIV4NormalUnpaired parametric two-tailed t test-40.41 to 38.080.9382  
    z5F, DIV7NormalUnpaired parametric two-tailed t test-125.3 to 133.20.9367  
    aa5G, DIV4NormalUnpaired parametric two-tailed t test-2.537 to 1.0950.3320  
    ab5G, DIV7NormalUnpaired parametric two-tailed t test-1.448 to 1.84640.7453  
    ac5H, DIV4NormalUnpaired parametric two-tailed t test-2.214 to 2.1610.9746  
    ad5H, DIV7NormalUnpaired parametric two-tailed t test-29.84 to 11.270.2780  
    ae5I, DIV4NormalUnpaired parametric two-tailed t test-0.2869 to 0.083550.2022  
    af5I, DIV7NormalUnpaired parametric two-tailed t test-0.1067 to 0.12670.8237  
    ag6D, DIV12NormalUnpaired parametric two-tailed t test-0.357 to 0.2460.6362  
    ah6D, DIV14NormalUnpaired parametric two-tailed t test-0.3671 to 0.086010.1601  
    ai6D, DIV16NormalUnpaired parametric two-tailed t test-0.3259 to 0.26870.8026  
    aj6E, DIV12NormalUnpaired parametric two-tailed t test-0.2159 to 0.46350.3689  
    ak6E, DIV14NormalUnpaired parametric two-tailed t test-1.22 to 0.8710.6674  
    al6E, DIV16NormalUnpaired parametric two-tailed t test-1.186 to 2.1160.4776  
    am6FNormalUnpaired parametric two-tailed t test-0.001536 to 0.16010.0537  
    an7ANormalUnpaired parametric two-tailed t test-2.46 to 5.5460.4326U 
    ao7DNongaussianMann–Whitney U test-13.7 to 17.23 (median)0.412029 
    ap7ENongaussianMann–Whitney U test-0.02334 to 0.1633 (median)0.235022.5 
    aq7FNongaussianMann–Whitney U test-0.2073 to 0.6074 (median)0.536228 
    ar7I (glutamate)NormalUnpaired parametric two-tailed t test-0.2091 to 0.33240.6538  
    as7I (glycine)NormalUnpaired parametric two-tailed t test-0.1241 to 0.18930.6822  
    at7I (KCl)NormalUnpaired parametric two-tailed t test-0.0722 to 0.50190.1416  
    au7I (NMDA)NormalUnpaired parametric two-tailed t test-0.1396 to 0.13860.9943  
    av7J (basal)NormalUnpaired parametric two-tailed t test-0.009 to 0.0080.9194  
    aw7J (stim)NormalUnpaired parametric two-tailed t test-0.219 to 0.0240.8919  
    ax7J (post)NormalUnpaired parametric two-tailed t test-0.018 to 0.0170.9343  
    ay7KNormalUnpaired parametric two-tailed t test-0.1198 to 0.02390.1375  
          rR 2
    az8ANormalPearson’s correlation-0.5771 to 0.00440.0535-0.31570.0997
          nadj p value
    ba8BNormalUnpaired parametric t test, multiple comparisons using Holm–SidakN/A0.0060
    -0.9991
    18 genes0.1026
    -0.9999
    bb8C, DIV4NormalUnpaired parametric two-tailed t test-2.717 to 2.1740.7735  
    bc8C, DIV7NormalUnpaired parametric two-tailed t test-2.219 to 3.4550.5780  
    bd8D, DIV4NormalUnpaired parametric two-tailed t test-6.521 to 7.7600.8215  
    be8D, DIV7NormalUnpaired parametric two-tailed t test-14.37 to 17.440.8017  
    bf8E, DIV4NormalUnpaired parametric two-tailed t test-61.79 to 156.90.2936  
    bg8E, DIV7NormalUnpaired parametric two-tailed t test-137.4 to 208.10.6001Fadj p value
    bh8HNormalOne-way ANOVA, Tukey’s post hoc-3.239 to 7.7740.5623(2,29) = 0.58730.5722
          H 
    bi8INongaussianKruskal–Wallis test, Dunn’s post hocN/A0.64330.88240.9999
    bj8JNongaussianKruskal–Wallis test, Dunn’s post hocN/A0.00949.3320.0071
    bk8KNongaussianKruskal–Wallis test, Dunn’s post hocN/A0.00709.9270.0343
          nadj p value
    bl9CNormalUnpaired parametric t test, multiple comparisons using Holm–SidakN/A0.0057
    -0.9251
    18 genes0.0976
    -0.9997
    bm9ENormalUnpaired parametric two-tailed t test-0.1507 to 0.43690.2783  
    bn9FNormalUnpaired parametric two-tailed t test-5.375 to 0.61410.0997  
    bo9GNormalUnpaired parametric two-tailed t test-48.93 to 136.50.2918  
    bp9INormalUnpaired parametric two-tailed t test-0.0644 to 0.58530.0943  
    bq9JNormalUnpaired parametric two-tailed t test-0.0166 to 0.12540.1297  
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High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons
Sara S. Parker, Aubin Moutal, Song Cai, Sambamurthy Chandrasekaran, Mackenzie R. Roman, Anita A. Koshy, Rajesh Khanna, Konrad E. Zinsmaier, Ghassan Mouneimne
eNeuro 13 September 2018, 5 (5) ENEURO.0135-18.2018; DOI: 10.1523/ENEURO.0135-18.2018

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High Fidelity Cryopreservation and Recovery of Primary Rodent Cortical Neurons
Sara S. Parker, Aubin Moutal, Song Cai, Sambamurthy Chandrasekaran, Mackenzie R. Roman, Anita A. Koshy, Rajesh Khanna, Konrad E. Zinsmaier, Ghassan Mouneimne
eNeuro 13 September 2018, 5 (5) ENEURO.0135-18.2018; DOI: 10.1523/ENEURO.0135-18.2018
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