Cell Viability Stain: One step, and ready-to-use. Allows for the rapid staining of dead cells by membrane penetration using propidium iodide.
DNA Standard for Flow Cytometry: Trout Red Blood Cell (TRBC) based DNA standard that is non-toxic and optimized for flow cytometry.
Nuclear Isolation Medium (NIM): Isolates intact nuclei from cells, tissues, frozen-thawed tissues, or deparafinized/enzymatically isolated tissues.
From the laboratory of Jerry T. Thornthwaite, PhD, Cancer Research Institute of West Tennessee.
Part of The Investigator's Annexe program.
|Product Type:||Buffer or Chemical|
|Name:||Cell Viability StainDNA Standard for Flow CytometryNuclear Isolation Medium (NIM)|
|Amount:||Cell Viability Stain: 10mL (100 reactions)DNA Standard for Flow Cytometry: 5mL of 1-2 x 106 TRBC/mL (100 reactions)Nuclear Isolation Medium (NIM): 100mL (100 reactions)|
Cell Viability Stain: Mix 100uL of Cell Viability Stain with 100uL of cells at 1x10^5-1x10^6 cells/mL)
DNA Standard for Flow Cytometry: Add 50uL of the DNA Standard to 1mL of cells at 1-2x10^6 cells/mL. Centrifuge to a pellet and resuspend in either NIM only (customized staining), NIM-DAPI, or NIM-PI. Alternatively, one may add portions of the DNA standard and cells directly to 1 mL of the NIM solutions and stain without centrifugation. The suspensions should be kept on ice or room temperature for 2-3 minutes before analyzing.
Nuclear Isolation Medium (NIM): In conjunction with the DNA Standard (see above), resuspend centrifuged pellet in 100uL of either NIM only (for customized staining), NIM-DAPI, or NIM-PI. Alternatively, one may add portions of the DNA standard and cells directly to 1 mL of the NIM solutions and stain without centrifugation. The suspensions should be kept on ice or room temperature for 2-3 minutes before analyzing.
Tonsil with TRBC DNA Standard and Nuclear Isolation Medium (NIM-DAPI)
The DNA Standard was mixed with NIM-DAPI, and filtered through a 37µm filter. The DNA standard served to establish the flow cytometer was operating within the precise boundaries of CV= 1-2%. Secondly, during a sample run, the DNA standard peak at channel 50 (approximately 5.0 pg/ nucleus). The position in channel 50 was used to assure the para enzymes were functioning by using the human tonsil to determine the optimum CV= 2-3% and the relative DNA value for the G0 tonsil nuclei of 7.4 pg/nucleus. Control paraffinated human tonsil tissues were also subjected to deparaffination and enzymatic dissociation to assure the PARA reagents were functioning properly. All tonsil controls were costhin a CV= 2-3% for each determination of DNA histogram measurements of the benign and cancer tissues. Also, the CV= 1-2% for all DNA standard samples. (A) Typical DNA Standard Trout Red Blood Cell (TRBC) Values. CV = 1.42 ± 0.19 SD, n=66. (B) Human Tonsil with DNA Standard. The data for 22 samples resulted in an average coefficient of variation of 2.18 ± 0.46 SD, an average DNA index of 1.42 ± 0.03 SD, and an average S-phase fraction of 4.37 ± 1.37 SD.
Adapted from: Thornthwaite, J.T., et al. J. Solid Tumors 3: 12-23 (2013).
If you publish research with this product, please let us know so we can cite your paper.