6 protocols
normalization data transformation protocol
The intensities of signals can be quantified by microarray scanner. The negative control was subtracted from the signal each sample, and when the value was less than 20 after the subtraction of negative control, the samples were eliminated from the list. Finally, the data was expressed as the relative signal level of cytokines (HSCT tumor per non-HSCT tumor).
array scanning protocol
The slides were blocked by incubation with the blocking buffer at room temperature for 30 minutes and incubated with the sample at room temperature for 1 hour. Slides were washed and incubated with biotin-conjugated antibodies at room temperature for 2 hours. Finally, the slides were washed and incubated with Alexa Flour 555-conjugated streptavidin at room temperature for 1 hour. The signals were detected using a microarray scanner (Innoscan; Brabrand, Denmark)
hybridization protocol
protocol see manufacturers website http://www.raybiotech.com/antibody-array.html
labeling protocol
protocol see manufacturers website http://www.raybiotech.com/antibody-array.html
nucleic acid extraction protocol
Collect tumors in eppendorf tubes . Lysed them with RIPA buffer (10 mMTris-HCI (pH 7.4), 1% deoxycholate, 1% NP40, 150 mM NaCl, 0.1% SDS, 0.2 mM phenylmethylsulfonyl fluoride, 1 microgram/ml aprotinin and 1 microgram/ml leupeptin) on ice for 20 min. Spin the tubes at 15,000 rpm x 5 min. Transfer all liquid part into eppendorf tubes.
treatment protocol
Nine-to-ten-week-old BALB/c mice received a lethal (9 Gy) irradiation on the day of transplantation. The irradiated mice were injected intravenously with bone marrow (BM) cells and splenic T cells from donor syngeneic mice. BM cells were isolated from donors by flushing each femur and tibia with RPMI-1640 medium supplemented with 5% FBS, and splenic cells were prepared by macerating the spleens. After lysis of the erythrocytes, splenic cells were incubated with anti-Thy-1.2 immunomagnetic beads (Miltenyi Biotec GmbH) at 4C for 15 min, followed by selection of T cells by AutoMACS (Miltenyi Biotec GmbH). CT26 cells were injected subcutaneously into the legs of the mice, and CT26 subcutaneous tumors were isolated 2 weeks later.