Description of the Resource
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The Immunology Core Laboratory (ICL) provides services in support of nonhuman primate research to investigators at the SNPRC and Texas Biomed, and to scientists from around the country. The ICL provides services in the areas of Flow Cytometry for cell sorting and phenotyping. Facility personnel are trained to assist researchers in flow cytometry experimental design, sample preparation techniques, and data analysis. Since 2013 the ICL has participated in two EQAPOL’s External Proficiency (EP) programs for Flow Cytometry; the EQAPOL (External Quality Assurance Oversight Laboratory) at Duke University is supported by the Division of AIDS, NIAID, and its goal is to assess factors contributing to variability in intracellular cytokine assays in human samples.
- FACS Aria.–The BD FACSAria™ III cell sorter is the first benchtop sorter that incorporates a fixed-alignment cuvette flow cell and can detect up to 18 colors, for a total of 20 parameters. This instrument was added to the laboratory in September of 2005, and upgraded with a fourth laser and an Automated Cell Deposition Unit (ACDU) in 2013. The addition of the ACDU allows the cell sorter to separate single cells in wells of a 6-, 12-, 24-, 48-, and 96-well plates.Cells passing through the cuvette flow cell are excited by four solid state lasers at 407-nm, 488-nm, 561-nm, and 633-nm.The light emitted by the 407 nm violet and 561 nm green lasers is detected with two octagons, each containing 8 photomultipliers (PMT); the light emitted by the 488 nm blue and 633 nm red lasesr is analyzed by two trigons with 3 PMT each.This cell sorter is contained within a Baker Bioprotect IV High Containment Biosafety cabinet within one of the BSL-3 laboratories. All sorting or analyses of unfixed cells, regardless of being exposed or not to bacterial or viral pathogens, are performed only in the FACS Aria.
- Cyan LX.–This instrument was obtained for the ICL in January of 2003.The CyAn™ LX Flow Cytometer provides three excitation lines, eleven parameters, complete compensation, and high-speed acquisition, in a bench-top configuration.The 405 nm, 488 nm, and 633 nm lasers are fixed and alignment-free, with spatially separated beams for minimal optical crosstalk. Eleven acquisition channels (including forward scatter, side scatter, and 9 colors) provide parallel processing of pulse height, pulse length, area, and log for each parameter.Logarithmic amplifiers for all channels provide a four log range in detection, with full intra- and inter-laser compensation of fluorescence spectral overlap.Analysis rates exceed 50,000 events per second.Variable, high-resolution sample control provides flow rates of 0 to 300 µl/second.
- 488 nm Excitation Reactive Dyes: FITC; PE; PE-TX Red; PE-Cy5, PerCP, PE-Cy5.5, PerCP-Cy5.5; PE-Cy7
- 635 nm Excitation Reactive Dyes: APC, Alexa Fluor 700, APC-Cy7, APC-H7
- 405 nm Excitation Reactive Dyes: Alexa Fluor 405, Pacific Blue, AmCyan, V500, BV510
For analysis of samples in the Cyan, samples need to be submitted in 5 ml polypropylene tubes (Falcon cat. # 352063) and fixed preferentially with 1% formaldehyde.
|Sample Preparation:||$55 per hour|
|Sample Acquisition (personnel):||$55 per hour|
|Sample Acquisition (material):||$0.25 per tube|
|Analysis:||$55 per hour|
|Cell Sorting:||$80 per hour|
|Cell Sorter Set-up Fee:||$50 per experiment|