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AccessionType
hybridization
Agilent One-Color Microarray-Based Gene Expression (Hybridization) Version: 5.7 Agilent publication number: G4140-90040 URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90040_One-Color_GE_5.7.pdf This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based one-color gene expression analysis.
labeling
P-AGIL-28
normalization data transformation protocol
The data were normalized using the percentile shift method to the 80th percentile.
array scanning protocol
Agilent SurePrint G3 mouse GE 8x60k microarray chips were scanned with an Agilent C Scanner with Surescan High Resolution Technology.
(Parameters: Scanning hardware = OTHER: Agilent C scanner, Scanning software = Scanning software)
nucleic acid extraction protocol
Cell pellets were resuspend in Trizol and frozen at -80oC. RNA was then extracted from the thawed samples via the phenol chloroform method.
(Parameters: Extracted product = total_RNA, Amplification = none)
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom+ infected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom+ infected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom+ infected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom- uninfected Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 12Hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom+ infected Kupffer cells.
growth protocol
3 C57BL/6 mice were injected with 200uL RPMI intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ Kupffer cells.
growth protocol
4 C57BL/6 mice were injected with 3x10e7 TdTom.LV9 amastigotes intravenously. 2hr later, the livers were removed, pooled and digested in with 0.4mg/mL warmed liberase TL (Roche) at 37oC for 45min, then homogenised. Digested livers were passed over 100uM cell strainers, washed in PBS containing 2%FCS and passed over a 33% percoll density gradient. The remaining cell pellet was washed, RBC lysed and the remaining cells labelled with F4/80, GR-1 and anti-CRIg and sorted on a MoFlo (DAKO) fluorescence activated cell sorter to isolate CRIg+ F4/80+ TdTom+ infected Kupffer cells.