Experiment Overview
| Repository ID: | FR-FCM-Z82B | Experiment name: | High-throughput flow cytometry analysis in yeast and human cells | MIFlowCyt score: | 82.27% |
| Primary researcher: | Maximilian Reuter | PI/manager: | Maximilian Reuter | Uploaded by: | Maximilian Reuter |
| Experiment dates: | 2023-01-01 - 2024-07-28 | Dataset uploaded: | Jul 2024 | Last updated: | Sep 2024 |
| Keywords: | [High Throughput] [DNA content] [EdU] [chromatin binding] [genome replication] [DNA replication] | Manuscripts: | [39241223] | ||
| Organizations: |
Institute of Molecular Biology (IMB) gGMBH , Mainz, RP (Germany)
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| Purpose: | Here, we present simple and advanced protocols for the analysis of total DNA content, de novo DNA synthesis, and protein association to chromatin in budding yeast and human cells. Upon optimization of experimental conditions and choice of fluorescent dyes, up to four parameters can be measured simultaneously and quantitatively for each cell of a population in a multi-well plate format. Reducing sample numbers, plastic waste, costs per well, and hands-on time without compromising signal quality or single-cell accuracy are the main advantages of the presented protocols. | ||||
| Conclusion: | Our protocols allow fast, high-throughput analyses of key replication parameters for both budding yeast and human cells in a 96-well plate format. We also present the first successful detection of yeast nuclear proteins by flow cytometry. A proof-of-principle application of antibody-based FLAG-tag detection allows qualitative and quantitative comparison of the association of various proteins to chromatin. Utilising our optimised protocols, we not only show that the central protein complexes of origin licensing, ORC and MCM2-7, are recruited in the same manner to yeast and human chromatin. We also provide measures to reduce hands-on time, reagent consumption, overall cost per well, and plastic waste. In addition, signal to noise ratios, throughput, and uniformity of protocols were optimised. Taken together, we provide a comprehensive tool kit for efficient and fast cytometric analysis of DNA replication parameters in cellula. | ||||
| Comments: | Our protocols try to minimize the ecological footprint of flow cytometry experiments by reducing hands-on time, reagent consumption, overall cost per well, and plastic waste. Please get in contact for template files and analysis files, as well as all files that cannot be downloaded. The database failed to upload some data properly. | ||||
| Funding: | This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation Project-ID: 505087959 and Project-ID: 393547839 – SFB 1361 | ||||
| Quality control: | Controls included: unstained samples, single stained samples, various antibodies coupled to different dyes, mutant strains, cell cycle arrests and biological replicates | ||||
Experiment variables
| Conditions | |
|---|---|
| · G1 arrested and released | Y8 75min click azide.fcs · Y8 15min click azide.fcs · Y8 30min click azide.fcs · Y8 45min click azide.fcs · Y8 60min click azide.fcs · Y8 90min click azide.fcs · Y8 G1 click azide.fcs · Y8 cyc click azide.fcs · Specimen1_E1_Y8 cyc.fcs · Specimen1_E2_Y8 G1.fcs · Specimen1_E3_Y8 15min.fcs · Specimen1_E4_Y8 30min.fcs · Specimen1_E5_Y8 45min.fcs · Specimen1_E6_Y8 60min.fcs · Specimen1_E7_Y8 75min.fcs · Specimen1_E8_Y8 90min.fcs · Fig3_MCM_1_105.fcs · Fig3_MCM_1_120.fcs · Fig3_MCM_1_15.fcs · Fig3_MCM_1_30.fcs · Fig3_MCM_1_45.fcs · Fig3_MCM_1_60.fcs · Fig3_MCM_1_75.fcs · Fig3_MCM_1_90.fcs · Fig3_MCM_1_G1.fcs · Fig3_MCM_1_cyc.fcs · Fig3_MCM_2_105.fcs · Fig3_MCM_2_120.fcs · Fig3_MCM_2_15.fcs · Fig3_MCM_2_30.fcs · Fig3_MCM_2_45.fcs · Fig3_MCM_2_60.fcs · Fig3_MCM_2_75.fcs · Fig3_MCM_2_90.fcs · Fig3_MCM_2_G1.fcs · Fig3_MCM_2_cyc.fcs · Fig3_MCM_3_105.fcs · Fig3_MCM_3_120.fcs · Fig3_MCM_3_15.fcs · Fig3_MCM_3_30.fcs · Fig3_MCM_3_45.fcs · Fig3_MCM_3_60.fcs · Fig3_MCM_3_75.fcs · Fig3_MCM_3_90.fcs · Fig3_MCM_3_G1.fcs · Fig3_MCM_3_cyc.fcs · Fig3_MCM_4_105.fcs · Fig3_MCM_4_120.fcs · Fig3_MCM_4_15.fcs · Fig3_MCM_4_30.fcs · Fig3_MCM_4_45.fcs · Fig3_MCM_4_60.fcs · Fig3_MCM_4_75.fcs · Fig3_MCM_4_90.fcs · Fig3_MCM_4_G1.fcs · Fig3_MCM_4_cyc.fcs · Specimen1_B2_Y8 G1 click picolyl.fcs · Specimen1_B5_Y8 45min click picolyl.fcs · Specimen1_B8_Y8 90min click picolyl.fcs · Specimen1_C2_Y8 G1 click.fcs · Specimen1_C5_Y8 45min.fcs · Specimen1_C8_Y8 90min.fcs · Sup6_D2_G1 Orc2 647.fcs · Sup6_D3_20min Orc2 647.fcs · Sup6_D4_40min Orc2 647.fcs · Sup6_D5_60min Orc2 647.fcs · Sup6_D6_80min Orc2 647.fcs · Sup6_E1_cyc Hta2 647.fcs · Sup6_E2_G1 Hta2 647.fcs · Sup6_E3_20min Hta2 647.fcs · Sup6_E4_40min Hta2 647.fcs · Sup6_E5_60min Hta2 647.fcs · Sup6_E6_80min Hta2 647.fcs · Sup6_cyc Orc2 647.fcs |
| · cdc6-1 permissive temperature | Specimen1_A1_C6_0_1.fcs · Specimen1_A2_C6_0_2.fcs · Specimen1_A3_C6_0_3.fcs · Specimen1_C1_C6_60_1_25.fcs · Specimen1_C2_C6_60_2_25.fcs · Specimen1_C3_C6_60_3_25.fcs · Specimen1_F1_C6_120_1_25.fcs · Specimen1_F2_C6_120_2_25.fcs · Specimen1_F3_C6_120_3_25.fcs |
| · cdc6-1 non-permissive temperature | Specimen1_B1_C6_30_1_37.fcs · Specimen1_B1_C6_30_1_37.fcs · Specimen1_B2_C6_30_2_37.fcs · Specimen1_B3_C6_30_3_37.fcs · Specimen1_D1_C6_60_1_37.fcs · Specimen1_D2_C6_60_2_37.fcs · Specimen1_D3_C6_60_3_37.fcs · Specimen1_E1_C6_90_1_37.fcs · Specimen1_E2_C6_90_2_37.fcs · Specimen1_E3_C6_90_3_37.fcs · Specimen1_G1_C6_120_1_37.fcs · Specimen1_G2_C6_120_2_37.fcs · Specimen1_G3_C6_120_3_37.fcs · Specimen1_H1_C6_150_1_37.fcs · Specimen1_H2_C6_150_2_37.fcs · Specimen1_H3_C6_150_3_37.fcs |
| · cdc46-1 permissive temperature | Specimen1_A4_M5_0_1.fcs · Specimen1_A4_M5_0_1.fcs · Specimen1_A5_M5_0_2.fcs · Specimen1_A5_M5_0_2.fcs · Specimen1_A6_M5_0_3.fcs · Specimen1_C4_M5_60_1_25.fcs · Specimen1_C5_M5_60_2_25.fcs · Specimen1_C6_M5_60_3_25.fcs · Specimen1_F4_M5_120_1_25.fcs · Specimen1_F5_M5_120_2_25.fcs · Specimen1_F6_M5_120_3_25.fcs |
| · cdc46-1 non-permissive temperature | Specimen1_B4_M5_30_1_37.fcs · Specimen1_B5_M5_30_2_37.fcs · Specimen1_B6_M5_30_3_37.fcs · Specimen1_D4_M5_60_1_37.fcs · Specimen1_D5_M5_60_2_37.fcs · Specimen1_D6_M5_60_3_37.fcs · Specimen1_E4_M5_90_1_37.fcs · Specimen1_E5_M5_90_2_37.fcs · Specimen1_E6_M5_90_3_37.fcs · Specimen1_G4_M5_120_1_37.fcs · Specimen1_G5_M5_120_2_37.fcs · Specimen1_G6_M5_120_3_37.fcs · Specimen1_H4_M5_150_1_37.fcs · Specimen1_H5_M5_150_2_37.fcs · Specimen1_H6_M5_150_3_37.fcs |
| · human cycling cells | Fig5 D_rep1.fcs · Fig5 D_rep2.fcs · Fig5E_MCM_2nd AB control.fcs · Fig5E_MCM_all.fcs · Fig5E_MCM_all2.fcs · Fig5E_MCM_click only.fcs · Fig5E_MCM_protein only.fcs · Fig5F_ORC_2nd AB only.fcs · Fig5F_ORC_all.fcs · Fig5F_ORC_all2.fcs · Fig5F_ORC_click only.fcs · Fig5F_ORC_protein only.fcs · HCT-cycling_MCM4.fcs · HCT_cycling_ORC2.fcs |
| · cycling cells yeast | Sup2_E1_xlinked.fcs · Sup2_full protocol.fcs · Sup2_osmotic shock.fcs · Sup2_spherplasts.fcs · Sup5C_G3_cyc_568.fcs · Sup5C_H3_cyc_647.fcs · Sup5C_cyc_405.fcs · Sup5C_cyc_PE.fcs |
| · human cells serum starvation | HCT116_2h serum release.fcs · HCT116_6h serum release.fcs · HCT116_Serum starvation.fcs |
| · human cells Nocodazole treated | HCT_Nocodazole arrest 12h_MCM4.fcs · HCT_Nocodazole arrest 9h_MCM4.fcs · HCT_Nocodazole arrest_12h ORC2.fcs · HCT_Nocodazole arrest_9h ORC2.fcs · HCT_Nocodazole arrest_MCM4.fcs · HCT_Nocodazole arrest_ORC2.fcs |
| · G1 arrested yeast cells | G1 2nd Ab_405.fcs · G1 2nd Ab_568.fcs · G1 2nd Ab_647.fcs · G1 2nd Ab_PE.fcs · G1 noAb_405.fcs · G1 noAb_568.fcs · G1 noAb_647.fcs · G1 noAb_PE.fcs |
| Sample Type | |
|---|---|
| · yeast | Y8 75min click azide.fcs · Y8 15min click azide.fcs · Y8 30min click azide.fcs · Y8 45min click azide.fcs · Y8 60min click azide.fcs · Y8 90min click azide.fcs · Y8 G1 click azide.fcs · Y8 cyc click azide.fcs · Specimen1_A1_C6_0_1.fcs · Specimen1_A2_C6_0_2.fcs · Specimen1_A3_C6_0_3.fcs · Specimen1_A4_M5_0_1.fcs · Specimen1_A4_M5_0_1.fcs · Specimen1_A5_M5_0_2.fcs · Specimen1_A5_M5_0_2.fcs · Specimen1_A6_M5_0_3.fcs · Specimen1_B1_C6_30_1_37.fcs · Specimen1_B1_C6_30_1_37.fcs · Specimen1_B2_C6_30_2_37.fcs · Specimen1_B3_C6_30_3_37.fcs · Specimen1_B4_M5_30_1_37.fcs · Specimen1_B5_M5_30_2_37.fcs · Specimen1_B6_M5_30_3_37.fcs · Specimen1_C1_C6_60_1_25.fcs · Specimen1_C2_C6_60_2_25.fcs · Specimen1_C3_C6_60_3_25.fcs · Specimen1_C4_M5_60_1_25.fcs · Specimen1_C5_M5_60_2_25.fcs · Specimen1_C6_M5_60_3_25.fcs · Specimen1_D1_C6_60_1_37.fcs · Specimen1_D2_C6_60_2_37.fcs · Specimen1_D3_C6_60_3_37.fcs · Specimen1_D4_M5_60_1_37.fcs · Specimen1_D5_M5_60_2_37.fcs · Specimen1_D6_M5_60_3_37.fcs · Specimen1_E1_C6_90_1_37.fcs · Specimen1_E2_C6_90_2_37.fcs · Specimen1_E3_C6_90_3_37.fcs · Specimen1_E4_M5_90_1_37.fcs · Specimen1_E5_M5_90_2_37.fcs · Specimen1_E6_M5_90_3_37.fcs · Specimen1_F1_C6_120_1_25.fcs · Specimen1_F2_C6_120_2_25.fcs · Specimen1_F3_C6_120_3_25.fcs · Specimen1_F4_M5_120_1_25.fcs · Specimen1_F5_M5_120_2_25.fcs · Specimen1_F6_M5_120_3_25.fcs · Specimen1_G1_C6_120_1_37.fcs · Specimen1_G2_C6_120_2_37.fcs · Specimen1_G3_C6_120_3_37.fcs · Specimen1_G4_M5_120_1_37.fcs · Specimen1_G5_M5_120_2_37.fcs · Specimen1_G6_M5_120_3_37.fcs · Specimen1_H1_C6_150_1_37.fcs · Specimen1_H2_C6_150_2_37.fcs · Specimen1_H3_C6_150_3_37.fcs · Specimen1_H4_M5_150_1_37.fcs · Specimen1_H5_M5_150_2_37.fcs · Specimen1_H6_M5_150_3_37.fcs · Specimen1_E1_Y8 cyc.fcs · Specimen1_E2_Y8 G1.fcs · Specimen1_E3_Y8 15min.fcs · Specimen1_E4_Y8 30min.fcs · Specimen1_E5_Y8 45min.fcs · Specimen1_E6_Y8 60min.fcs · Specimen1_E7_Y8 75min.fcs · Specimen1_E8_Y8 90min.fcs · Fig3_MCM_1_105.fcs · Fig3_MCM_1_120.fcs · Fig3_MCM_1_15.fcs · Fig3_MCM_1_30.fcs · Fig3_MCM_1_45.fcs · Fig3_MCM_1_60.fcs · Fig3_MCM_1_75.fcs · Fig3_MCM_1_90.fcs · Fig3_MCM_1_G1.fcs · Fig3_MCM_1_cyc.fcs · Fig3_MCM_2_105.fcs · Fig3_MCM_2_120.fcs · Fig3_MCM_2_15.fcs · Fig3_MCM_2_30.fcs · Fig3_MCM_2_45.fcs · Fig3_MCM_2_60.fcs · Fig3_MCM_2_75.fcs · Fig3_MCM_2_90.fcs · Fig3_MCM_2_G1.fcs · Fig3_MCM_2_cyc.fcs · Fig3_MCM_3_105.fcs · Fig3_MCM_3_120.fcs · Fig3_MCM_3_15.fcs · Fig3_MCM_3_30.fcs · Fig3_MCM_3_45.fcs · Fig3_MCM_3_60.fcs · Fig3_MCM_3_75.fcs · Fig3_MCM_3_90.fcs · Fig3_MCM_3_G1.fcs · Fig3_MCM_3_cyc.fcs · Fig3_MCM_4_105.fcs · Fig3_MCM_4_120.fcs · Fig3_MCM_4_15.fcs · Fig3_MCM_4_30.fcs · Fig3_MCM_4_45.fcs · Fig3_MCM_4_60.fcs · Fig3_MCM_4_75.fcs · Fig3_MCM_4_90.fcs · Fig3_MCM_4_G1.fcs · Fig3_MCM_4_cyc.fcs · G1 2nd Ab_405.fcs · G1 2nd Ab_568.fcs · G1 2nd Ab_647.fcs · G1 2nd Ab_PE.fcs · G1 noAb_405.fcs · G1 noAb_568.fcs · G1 noAb_647.fcs · G1 noAb_PE.fcs · Specimen1_B2_Y8 G1 click picolyl.fcs · Specimen1_B5_Y8 45min click picolyl.fcs · Specimen1_B8_Y8 90min click picolyl.fcs · Specimen1_C2_Y8 G1 click.fcs · Specimen1_C5_Y8 45min.fcs · Specimen1_C8_Y8 90min.fcs · Sup2_E1_xlinked.fcs · Sup2_full protocol.fcs · Sup2_osmotic shock.fcs · Sup2_spherplasts.fcs · Sup5C_G3_cyc_568.fcs · Sup5C_H3_cyc_647.fcs · Sup5C_cyc_405.fcs · Sup5C_cyc_PE.fcs · Sup6_D2_G1 Orc2 647.fcs · Sup6_D3_20min Orc2 647.fcs · Sup6_D4_40min Orc2 647.fcs · Sup6_D5_60min Orc2 647.fcs · Sup6_D6_80min Orc2 647.fcs · Sup6_E1_cyc Hta2 647.fcs · Sup6_E2_G1 Hta2 647.fcs · Sup6_E3_20min Hta2 647.fcs · Sup6_E4_40min Hta2 647.fcs · Sup6_E5_60min Hta2 647.fcs · Sup6_E6_80min Hta2 647.fcs · Sup6_cyc Orc2 647.fcs |
| · human | Fig5 D_rep1.fcs · Fig5 D_rep2.fcs · Fig5E_MCM_2nd AB control.fcs · Fig5E_MCM_all.fcs · Fig5E_MCM_all2.fcs · Fig5E_MCM_click only.fcs · Fig5E_MCM_protein only.fcs · Fig5F_ORC_2nd AB only.fcs · Fig5F_ORC_all.fcs · Fig5F_ORC_all2.fcs · Fig5F_ORC_click only.fcs · Fig5F_ORC_protein only.fcs · HCT-cycling_MCM4.fcs · HCT116_2h serum release.fcs · HCT116_6h serum release.fcs · HCT116_Serum starvation.fcs · HCT_Nocodazole arrest 12h_MCM4.fcs · HCT_Nocodazole arrest 9h_MCM4.fcs · HCT_Nocodazole arrest_12h ORC2.fcs · HCT_Nocodazole arrest_9h ORC2.fcs · HCT_Nocodazole arrest_MCM4.fcs · HCT_Nocodazole arrest_ORC2.fcs · HCT_cycling_ORC2.fcs |
