Sex as a Biological Variable
We used female NOD scid mice for our xenograft experiments. Our previous experience demonstrated that sex has no effects on these experiments.
Cell Cultures
Rat1 fibroblasts, human epithelial cell ARPE-19, human colon cancer cell DLD1, and human glioblastoma cells GBM9, SF188, U87 and LN229 were cultured in DMEM with 5% FBS, 100 U/mL pen/strep. For suspension culture, LN229 cells were cultured in DMEM with 5% FBS, 100 U/mL pen/strep on plate coated with poly-HEMA. To induce differentiation, spheres were cultured in DMEM/F12 + B27 + 0.5 ml/ml BSA + 20 ng/ml EGF + 20 ng/ml FGFb for 10 days. Human-induced pluripotent stem cells (iPSCs) with doxycycline-inducible neurogenin-2 (NGN2), gifted from Dr. Erik Ullian (UCSF), were cultured in Essential 8 (E8) medium. Neuronal differentiation was induced using induction medium (DMEM/F12, HEPES + N2 Supplement + Non-essential Amino Acids + L-Glutamine + 2 mg/ml doxycycline) for 3 days 14. siRNA was reverse transfected to cells with Lipofectamine RNAiMAX reagent following the standard protocol (Invitrogen). Briefly, on the day of transfection, 2.5 x 105 cells were seeded per well in 6-well plates and transfected with 5 µL siRNA (20 µM) and 5 µL Lipofectamine previously mixed in Opti-MEM for 15 min. After 72 h of transfection, cells were collected and analyzed.
Stable cell lines
ARNT2 knockout (KO) cells were generated by transfecting ARNT2 CRISPR/Cas9 KO Plasmid (Santa Cruz sc-403101) into LN229 cells with Lipofectamine LTX&Plus reagent. Three days after transfection, GFP-positive cells were sorted by flow cytometry and single clones were selected to verify the knockout by using Western blots. For cell lines stably expressing ARNT2, the ARNT2 gene was constructed into pIRESpuro vector with N-terminal GFP tag, and the construct, together with the empty vector, were transfected into LN229 cells. The cells that stably expressing these constructs were selected with 5 mg/mL puromycin. The ARNT2 gene was also constructed into pIRESneo vector with no tag. The construct, together with the empty vector, were transfected into GBM9 cells. The cells stably expressing these constructs were selected with 1 mg/mL G418.
Cell Viability Assays
Cell proliferation was measured by either crystal violet staining or Cell Counting Kit-8 (CCK-8). For crystal violet staining, the cells were cultured in 6-well or 12-well plates for 3-7 days before washed with PBS, and then fixed with methanol at room temperature (RT) for 10 min. Cells were stained with crystal violet solution containing 1% acetic acid, 1% methanol, 1% (w:v) crystal violet dye for 10 min with agitation. After washing extensively, the plates were scanned and the intensity of the signal was quantitated by using ImageJ. Results are presented to reflect the relative growth after normalization by the control condition. Experiments were repeated at least three times.
For analysis via Cell Counting Kit-8 (CCK-8), the cells were cultured in 96-well plates for 3-7 days. After the addition of 10 mL of CCK-8 reagent, the cells were put back into the incubator for 1 h and absorbance was read at 450 nm. Results are presented to reflect the relative growth after normalization by the control condition. Experiments were repeated at least three times.
Immunofluorescence Staining and Microscopic Imaging
Cells grown on glass plates were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT), and permeabilized in blocking buffer (0.1% Saponin, 3% BSA in PBS) for 20 min at 4°C. Primary antibodies in blocking buffer were added and incubated 1 hour at RT. Cells were washed 3 times with 0.1% Saponin in PBS and incubated with secondary antibodies in blocking buffer for 1 hour at RT. DAPI was used to stain the nuclei. Images were acquired with a Zeiss LSM780 microscope.
Western Blots of Total Cell Lysates and Nuclear/cytoplasmic Fractionation
Total protein was extracted with lysis buffer containing 10 mM Tris-HCl (pH 7.7), 50 mM NaCl, 0.5% Nonidet P-40, proteinase inhibitor cocktail (SIGMAFAST™ Protease Inhibitor Cocktail Tablets, EDTA-Free), and 1 mM DTT. For fractionation, the cells were extracted with Buffer A (10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5% Nonidet P-40) plus proteinase inhibitor cocktail and 1 mM DTT by vortex. The samples were spun at 4,000 rpm for 4 min, and the supernatants were collected as cytoplasmic fractions. The pellets were washed twice with Buffer A and then extracted with Buffer B (20 mM HEPES-KOH pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 15% glycerol) plus proteinase inhibitor cocktail and 1 mM DTT with sonication. The samples were spun at 15,000 rpm for 10 min, and the supernatants were collected as nuclear fraction. Protein concentrations were measured by using the Bradford protein assay, and the samples were normalized to same protein concentration. Proteins were separated by SDS–polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Thermo Fisher), probed with specific antibodies, and detected by using chemiluminescence with the Bio-Rad Image lab.
RNA-seq
WT and ANRT2 KO LN229 cells were collected and sent to GENEWIZ for RNA extraction and sequencing. RNA-seq assessment of the raw sequencing reads was done using the NGS-QC-Toolkit 36. The reads were aligned to the genome RGSC 6.0/rn6 using HISAT2 (v 2.1.0) aligner. A minimum read count filter of 10 total reads was applied to remove low-expressed genes. Filtered reads were then normalized using DESeq2. DeSeq2 employs a negative binomial distribution to estimate data variability and uses an error model for a more robust statistical test for significance. An FDR cutoff of <5% was used to select significantly altered genes between experiment conditions. Gene Ontology analysis was performed with GO Consortium (http://geneontology.org/). RNA-seq data has been deposited in GEO with ID GSE236486.
RT-qPCR
RNA was extracted from cells with QIAGEN RNeasy Mini Kit and reverse transcribed to cDNA with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). Relative RNA levels were measured by qPCR with iTaq™ Universal SYBR® Green Supermix (Bio-Rad) and the Bio-Rad CFX96 device and normalized to 18S rRNA gene.
Bioinformatics Analysis
ARNT2 mRNA expression in tumor and normal tissues from patients of different tumor types deposited in TCGA and GTEx was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) web server. |Log2FC| cutoff 0.585, and p-value cutoff 0.01. Kaplan-Meier curve comparing survival of cancer patients with the 25% highest and the 25% lowest levels of certain genes were generated using Xena Functional Genomics Explorer (Xena Functional Genomics Explorer). Gene expression levels in glioma grades were from TCGA and Chinese Glioma Genome Atlas (CGGA) (http://www.cgga.org.cn/).
Subcutaneous Xenografts
LN229 or GBM9 cells suspended in 30% Matrigel were injected into the flank of each female NOD scid mouse (Jackson lab). Mice were sacrificed when their largest tumors reached ~2 cm. At the end of the experiment, tumors were harvested, weighed, and either snap frozen in liquid nitrogen for protein extraction or fixed with 10% formalin for histology. All procedures are approved by IACUC at UT Southwestern Medical Center.
Orthotopic Xenograft
GBM9 cells suspended in HBSS (Ca-, Mg-) were injected orthotopically into the striatum of 9 weeks old NOD scid mice using the following coordinates (AP, 0 mm; ML, –2.5 mm; DV, –3 mm) using bregma as the starting cranial landmark (Note: While the injection took place at a depth of -3 mm in the dorsal/ventricle plane cells were injected at -2.5 mm in the dorsal/ventricle plane to allow for a 0.5 mm gap for cells to accumulate in.). Cells were injected at a rate of 1uL/20 seconds over one minute, followed by a 30-second hold before removing the syringe. All injections were facilitated through the use of a stereotactic frame. Mice were monitored weekly using bioluminescent imaging to confirm tumor engraftment/progression. Mice were euthanized based on co-morbidities, including weight, seizures, hunched back, matted fur, and head swelling.
Histology
Tumor tissues from xenografts experiments were fixed with 10% neutral buffered formalin for 2 days and then transferred to 70% ethanol before hematoxylin and eosin (H&E) staining performed by the UTSW tissue core.
Metabolomics Analyses
20-25 mg xenograft tumor tissues were homogenized and extracted with 300 ml of ice-cold methanol/water 80:20 (vol/vol). Dry samples equivalent to 10 mg of protein with SpeedVac. Qualitative assessment of global metabolites by mass spectrometry were performed by the metabolomics facility at the Children’s Research Institute (UT Southwestern Medical Center, UTSW). Using a cutoff of 30% changes and p value under 0.1.
Quantification and Statistical Analysis
All statistical analyses were performed using two-tailed student T-test. P <0.05 was considered statistically significant. All values are reported as mean ± SEM in each figure.
RESOURCES TABLE
Cell lines
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|
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Reagent or Resource
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Source
|
Identifier
|
Rat1 fibroblasts
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Dr. John Sedivy (Brown University)
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N/A
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ARPE-19
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Sandra Schmid lab
|
N/A
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DLD1
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ATCC
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CCL-221
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LN229
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ATCC
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CCL-2611
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U-87 MG
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ATCC
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HTB-14
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GBM9
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Dr. Sandeep Burhma (UT, San Antonio)
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SF188
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Sigma
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SCC282
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|
|
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Plasmids
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Reagent or Resource
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Source
|
Identifier
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ARNT2 CRISPR/Cas9 KO Plasmid (h)
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Santa Cruz
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sc-403101
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pIRESpuro-GFP-ARNT2
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This study
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N/A
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pIRESneo-ARNT2
|
This study
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N/A
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|
|
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Antibodies
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|
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Reagent or Resource
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Source
|
Identifier
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ARNT2
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Proteintech
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12810-1-AP
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MYC
|
Abcam
|
ab32072
|
AHR
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Enzo Life Sciences
|
BMLSA2100100
|
ARNT
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Novus Biologic
|
NB100-110
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NPAS4
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Sigma
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SAB1402073
|
SIM2
|
MYBIOSOURCE
|
MBS129140
|
BMAL
|
Cell Signaling
|
14020
|
CLOCK
|
Cell Signaling
|
5157
|
EZH1
|
Cell Signaling
|
42088
|
EZH2
|
Cell Signaling
|
5246
|
SUZ12
|
Cell Signaling
|
3737
|
Histone H3
|
Cell Signaling
|
4499
|
Histone H3 (tri methyl K27)
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Abcam
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ab6002
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AHRR
|
Abcam
|
ab108518
|
MAX
|
Santa Cruz
|
sc-197
|
MNT
|
Santa Cruz
|
sc-769
|
MYCN
|
Santa Cruz
|
SC-142
|
Nestin
|
Santa Cruz
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sc-23927
|
GFAP
|
Cell Signaling
|
12389
|
Acetylated Tubulin
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Sigma
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T7451
|
α-Tubulin
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Sigma
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T6199
|
Phospho-p70 S6 Kinase (Thr389)
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Cell Signaling
|
9234
|
p70 S6 Kinase
|
Cell Signaling
|
2708
|
Phospho-mTOR (Ser2448)
|
Cell Signaling
|
5536
|
mTOR
|
Cell Signaling
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2983
|
Cyclin A1
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Novus Biologic
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MAB7046
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p27 Kip1
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Cell Signaling
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3686
|
β3-Tubulin
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Cell Signaling
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5568
|
UCHL1
|
Cell Signaling
|
13179
|
Sox2
|
Cell Signaling
|
3579
|
Oct-4A
|
Cell Signaling
|
2840
|
Neurofilament-L
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Cell Signaling
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2837
|
Synaptophysin
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Cell Signaling
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36406
|
β-Actin
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Cell Signaling
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4970
|
|
|
|
Chemicals
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|
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Reagent or Resource
|
Source
|
Identifier
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UNC1999
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Sigma
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5050520001
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GSK126
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Sigma
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5005800001
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|
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Oligonucleotides for qPCR
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|
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Gene
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F sequence
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R sequence
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Human ARNT2
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gcctatcctctccgagca
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ccaggtatgtctgaagccattt
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Human MYC
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caccagcagcgactctga
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gatccagactctgaccttttgc
|
Rat ARNT2
|
gaggatcagagccacctacg
|
gcacttggcccttcagttta
|
Rat MYC
|
gtgctgcatgaagagacacc
|
tcaatttcttcctcatcatcttgt
|
Rat AHR
|
cttcagatgccggctgag
|
cctcccttggaaattcattg
|
Rat ARNT
|
ccactgcacaggttacatcaa
|
tcatcatctgggagggagac
|
18S rRNA
|
accgcagctaggaataatgga
|
gcctcagttccgaaaacca
|
|
|
|
siRNA oligonucleotides
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|
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Reagent or Resource
|
Source
|
Identifier
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Universal controls #2
|
Sigma mission
|
SIC002
|
siARNT2-1
|
Sigma Mission
|
SASI_Hs01_00078252
|
siARNT2-2
|
Sigma Mission
|
SASI_Hs01_00078253
|
siARNT2-3
|
Sigma Mission
|
SASI_Hs01_00078254
|
siARNT2-4
|
Sigma Mission
|
SASI_Hs02_00346447
|
siMYC-3
|
Sigma Mission
|
SASI_Hs01_00222678
|
siMYC-4
|
Sigma Mission
|
SASI_Hs01_00222680
|
siEZH1
|
Sigma Mission
|
SASI_Hs02_00332978
|
siEZH2
|
Sigma Mission
|
SASI_Hs01_00078252
|
siSUZ12-1
|
Sigma Mission
|
SASI_Hs02_00347682
|
siSUZ12-2
|
Sigma Mission
|
SASI_Hs01_00107270
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siAHR-1
|
Sigma Mission
|
SASI_Hs02_00332181
|
siAHR-2
|
Sigma Mission
|
SASI_Hs01_00140198
|
siAHR-4
|
Sigma Mission
|
SASI_Hs01_00140199
|
siAHRR-1
|
GUU UGG AAG UAC AGA GAA A
|
N/A
|
siAHRR-2
|
CAG CAA CGA UCG UGG ACU A
|
N/A
|
siAHRR-3
|
UCA AAU AUA AGG UGG GAA U
|
N/A
|
siAHRR-4
|
GGG ACA AGA CAG ACA AGU A
|
N/A
|
siMLX-1
|
Sigma Mission
|
SASI_Hs01_00163686
|
siMLX-2
|
Sigma Mission
|
SASI_Hs01_00163687
|
siMAX-1
|
Sigma Mission
|
SASI_Hs01_00011941
|
siMAX-3
|
Sigma Mission
|
SASI_Hs01_00011942
|
siMNT-1
|
Sigma Mission
|
SASI_Hs02_00353224
|
siMNT-2
|
Sigma Mission
|
SASI_Hs01_00094368
|
|
|
|
Databases
|
|
|
Reagent or Resource
|
Source
|
Identifier
|
RNA-seq Rat1 fibroblasts MYC OE
|
Maralice Conacci-Sorrell lab
|
Lafita-Navarro et al., 2018
|
RNA-seq LN229 ARNT2 KO
|
Maralice Conacci-Sorrell lab
|
GSE236486
|
|
|
|
Software
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|
|
Reagent or Resource
|
Source
|
Identifier
|
FIJI
|
Image J software
|
N/A
|
Prism 10
|
GraphPad
|
N/A
|
|
|
|
Other
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|
|
Reagent or Resource
|
Source
|
Identifier
|
ChemiDoc Imaging System
|
BIO-RAD
|
17001401
|
CFX96 Touch Real-Time PCR Detection System
|
BIO-RAD
|
1855195
|
Zeiss LSM780 Inverted confocal microscope
|
Zeiss
|
N/A
|
|
|
|