Fig 1.
Schematic of cell separation techniques.
A) Positive selection of astrocytes was performed using an ACSA-2 antibody. Dissociated cells were incubated with magnetic beads conjugated with an ACSA-2 antibody, and were applied to a ferromagnetic column placed in a magnetic field. The astrocyte enriched cell population was eluted from the column after successive washes that removed non-target cell populations. B) Neurons were enriched using a negative selection protocol. Dissociated cells were incubated with biotin-conjugated antibodies targeting non-neuronal cell populations, followed by labelling with streptavidin-coated magnetic beads. Neuronal cell populations were eluted, while biotin-bound cells were retained on the magnetic column. C) Positive selection of cells is customizable. Here an antibody which detects an extracellular epitope of GLT-1 was used to enrich astrocytes. Dissociated cells were labeled with the rabbit-anti-GLT-1 antibody and subsequently labeled with anti-rabbit microbeads. The enriched population was retained on the column, and eluted subsequent to washing undesired cells from the magnetic column.
Fig 2.
Isolation of astrocytes across development.
Astrocytes were isolated using an ACSA-2 antibody conjugated to magnetic microbeads. A, B, C) Quantitative PCR data analysis of isolated astrocytes as demonstrated by expression of the astrocytic genes SLC1A3, SLC1A2, and GFAP in young (p7-p10), juvenile (p20-p30), and adult (p60-p80) mice. In contrast, there are low levels of expression of oligodendrocyte gene MBP, microglia gene ITGAM, and neuronal gene RBFOX3. D, E) The efficiency of the ACSA-2 positive selection technique is indicated by qPCR data which demonstrates depletion of astrocytic genes SLC1A3 and SLC1A2 in the microglia and myelin debris removal fraction as well as the non-targeted cell population or “flow through” (example shown from p20-p30 isolation). F) Quantitative PCR data analysis of isolated astrocytes without addition of “clean up” step demonstrates minimal contamination of microglia and oligodendrocyte genes, ITGAM and MBP, respectively (example shown from p20-p30 isolation). G) Quantitative PCR data analysis of flow through fraction from experiments omitting the microglia and myelin debris removal step demonstrates low expression of astrocyte gene expression. H) Chromatograph demonstrating RNA integrity isolated from enriched population. RNA isolated from an adult animal (p70s) was Dnase treated and analyzed for quality. For all time points qPCR data is represented as mean +/- SEM with a minimum of n = 3. The mean differences between relative gene expression were analyzed by ANOVA, *p < 0.05.
Fig 3.
Isolation of neurons across development.
A, B, C) Quantitative PCR analysis demonstrates isolation of neurons in young (p7-p10), juvenile (p20-p30), and adult (p60-p80) mice. Low expression of astrocytic gene SLC1A3, microglial gene ITGAM, and oligodendrocyte gene MBP was found with concomitant high expression of neuronal genes RBFOX3 and NEFM. Data from qPCR is represented as mean +/- SEM with a minimum of n = 3 for all time points. The mean differences between relative gene expression were analyzed by ANOVA, *p < 0.05.
Fig 4.
Customized isolation of astrocytes and microglia.
Astrocytes were enriched using an antibody which recognizes an extracellular epitope of glutamate transporter GLT-1. A, B, C) Quantitative PCR analysis of the enriched astrocyte population in young (p7-10), juvenile (p20-p30), and adult (p60-p80) mice demonstrates enrichment of astrocytic genes SLC1A3, SLC1A2, and GFAP with low expression of oligodendrocyte and neuronal genes. C) Microglia contamination was seen in isolated populations from adult (p60-80) mice as indicated by moderate expression of microglia gene ITGAM. D) The microglia contamination was resolved by removing microglia with Cd11b+ microbeads prior to isolating astrocytes (example shown from p60-p80 isolation), as demonstrated by the minimal oligodendrocyte, microglia, and neuronal gene expression. E) Concomitantly, the microglia removal step allowed for the enrichment of Cd11b+ microglia in adult (p60) animals. Low expression of astrocytic, oligodendrocyte, and neuronal genes was found in the Cd11b+ isolated fraction. Data from qPCR is represented as mean +/- SEM with a minimum of n = 3 for all experiments. The mean differences between relative gene expression were analyzed by ANOVA, *p < 0.05.
Fig 5.
Increased retention of processes with magnetic separation.
A) Analysis of the positive selection of astrocytes from rat cortex demonstrates a depletion of oligodendrocyte, microglia, and neuronal marker genes (MBP, ITGAM, and MAP2, respectively) with high expression of astrocytic gene expression, SL1CA2 and GFAP, n = 6. The mean differences between relative gene expression were analyzed by ANOVA, * p < 0.05. B) Representative images of S100β eGFP positive astrocytes from i) pre-sorting, ii) post-magnetic sorting, and iii) post-FAC sorting of rat cortex. Images demonstrate that magnetic sorting resulted in an increased number of cells retaining complexity post-sorting. C) Shape Index quantification of cellular complexity demonstrates that magnetic sorting leads to an increased cellular complexity over FAC sorted cells. Mean Shape Index differences were analyzed by student’s t-test, *p < 0.001
Table 1.
Genes and proteins used to distinguish cellular populations.
Fig 6.
Protein expression in isolated populations.
Following isolation, proteins were extracted and immunoblotting used to determine protein expression relative to whole cortex homogenates. A) Western blot analysis of proteins from magnetically enriched astrocyte populations in rat cortex demonstrates not only an increase in astrocytic protein expression, but also an increase in extrasomatic protein expression. B) Western blot analysis of proteins from magnetically separated cortical astrocyte populations in young, juvenile, and adult mice demonstrates similar trends in protein expression. Astrocyte-specific protein expression was enriched in the separated populations. For each time point, only one example is shown. C) Western blot analysis of magnetically separated neuronal populations in mice across developmental time points. Neuronal-specific protein expression was observed in the separated populations. D) Western blot analysis of microglia enriched populations following magnetic separations across development. Enrichment of microglia protein expression was observed, concomitant with depletion of non-specific primary antibody. For each time point, one experiment is shown.