Multiparameter FACS assays to detect alterations in cellular parameters and screening
A cell parameter, cell technology, applied in the field of peptide and other chemical libraries, combinatorial chemical libraries, can solve the problem that a single receptor blocker cannot overcome the influence and so on
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Embodiment 1
[0194] Cell cycle analysis using p21 as a positive control Materials and methods:
[0195] Vector construction: The coding region of the p21 gene was cloned from Jurkat cDNA by PCR with upstream primers covering the starting methionine (5'-GATCGGATCCACCACCATGGGCTC AGAACCGGCTGGGGATGTC) and the C-terminus (5'-GATCCCAATTTAATGGTTTTATTT GTCATCGTCATCCTTGTAGTCGGGCTTCCTCTTGGAGAAGATCAGCCGGCGTTTG). A single PCR product was directional cloned into the CRU5-GFP retroviral vector (Rigel Company) through the flanking BstX1 site in the primer. The resulting construct, CRU5-GFP-p21F ( figure 1 ), encoding GFP fused (in-frame) to the human p21 protein with a Gly inserted at position 2, with a FLAG-epitope at the C-terminus. 将p21的C-末端24个氨基酸通过PCR引物中的旁侧BstX1位点克隆到GRU5-GFP逆转录病毒载体(Rigel公司)中,所述PCR引物为:5′GATCCCACCACCATGGGCAAACGGCGGCAGACCAGCATGACAGATTTCTACCACTCCAAACGCC GGCTGATCTTCTCCAA; 5′GATCCCAATTTAAATGGTTTTATTTGTCATCGTCATCCTTGTAGTCGGGCTTCCTCTTGGAGAAG ATCAGCCGGCGTTTG. The resulting cons...
Embodiment 2
[0201] Population-Based Exocytase Activity Assay
[0202] Materials: All chemical reagents were obtained from Sigma Chemical Company. Dyes and glucuronides were obtained from Molecular Probes. Cell lines MC-9 and RBL-2H3 were obtained from the American Type Culture Collection (ATCC). Cell culture reagents were obtained from Fisher Scientific, and molecular biology reagents were obtained from Clontech.
[0203] Cell culture: MC-9 cells were loaded with L-arginine (116mg / ml), L-aspartic acid (36mg / ml), sodium pyruvate (1mM), non-essential amino acids (0.1mM), Folic acid (6mg / ml), 2-mercaptoethanol (0.05mM), L-glutamine (2mM), heat-inactivated fetal bovine serum (10%) in DMEM, and 10% T-stim conditioned medium (Collaborative Research , Inc.) as suspension cultures in shake flasks. Cell densities were maintained at 0.25 and 2 x 106 / ml. Experiments were performed on cells with greater than 95% cell viability as determined by trypan blue exclusion. Make RBL-2H3 c...
Embodiment 3
[0208] Example 3: Changes in Light Scattering of Mast Cell Exocytosis
[0209] Cells were prepared as described in Example 2, and light scattering properties were determined.
[0210] Results: The results are shown in Figure 4. Changes in light scatter (side scatter versus front scatter) observed by flow cytometry of RBL-2H3 cells (A, D) and MC-9 cells (B, C, E, F) are plotted as bivariate histograms. Cells were stimulated with ionophore A23187 (0.5 μg / ml) and observed at different time points [0 min (A,C), 5 min (E), 10 min (D), and 30 min (B,F)]. Time-dependent scatter changes were evident, with both cell lines changing significantly within the first 10 min, representing bulky exocytate in these cells.
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