Experiment Overview
| Repository ID: | FR-FCM-Z7CU | Experiment name: | NKG2D overexpression by mRNA transfection with CART ONA in human primary NK cells | MIFlowCyt score: | 97.00% |
| Primary researcher: | Ruoxi Pi | PI/manager: | Catherine Blish | Uploaded by: | Ruoxi Pi |
| Experiment dates: | 2024-01-22 - 2024-01-23 | Dataset uploaded: | Apr 2024 | Last updated: | Oct 2025 |
| Keywords: | [NKG2D] [charge-altering releasable transporters (CART)] [natural killer (NK) cells] | Manuscripts: | |||
| Organizations: |
Stanford University, Department of Medicine, Division of Infectious Diseases, Palo Alto, California (USA)
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| Purpose: | We are investigating the function of natural killer (NK) cell receptor, NKG2D, in the functional response of NK cells to HIV-infected autologous CD4+ T cells. We will overexpress NKG2D in human primary NK cells by transfecting them with charge-altering releasable transporters (CARTs) to deliver the mRNA that encodes NKG2D. DAP10 is the natural signaling adaptor of NKG2D in human NK cells, and co-transfection of DAP10 can enhance the expression of exogenous NKG2D on the surface of NK cells. In this experiment, we are testing which ratio of co-transfecting NKG2D and DAP10 mRNA allows the highest expression level of NKG2D on the surface of the transfected NK cells. To distinguish NK cells that have translated the exogenous NKG2D mRNA from the ones that naturally express NKG2D, we will co-transfect NK cells with GFP mRNA and GFP expression will indicate the transfected NK cells. | ||||
| Conclusion: | We tested co-transfection of NK cells with NKG2D+DAP10+GFP mRNA at 2:1:2, 3:1:3, 4:1:4, and 1:1 (1:1 ratio of NKG2D:GFP, no DAP10) mass ratios. Among all the four ratios tested, we identified that 3:1:3 allowed slightly higher expression level of NKG2D in GFP+ cells, compared to 2:1:2 and 4:1:4, indicated by the mean fluorescent intensity of NKG2D staining. When no DAP10 mRNA was used for co-transfecting NK cells, there is no considerable increase of NKG2D expression in GFP+ cells compared to GFP- NK cells, suggesting that exogenous DAP10 is required for the surface expression of the exogenous NKG2D. | ||||
| Comments: | In this experiment, we tested the co-transfection of NKG2D+DAP10+GFP mRNA at 4 different mass ratios. 4:1:4 means that 44.4 ng of NKG2D mRNA, 11.1 ng of DAP10 mRNA and 44.4 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 3:1:3 means that 42.9 ng of NKG2D mRNA, 14.3 ng of DAP10 mRNA and 42.9 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 2:1:2 means that 40 ng of NKG2D mRNA, 20 ng of DAP10 mRNA and 40 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. 1:1 means that 50 ng of NKG2D mRNA and 50 ng of GFP mRNA were mixed and complexed with CART ONA and added into 500,000 NK cells for transfection. The expression of NKG2D and GFP was analyzed 16 hours after transfection. | ||||
| Funding: | This work was supported by the National Institutes of Health (NIH R01 AI161803). | ||||
| Quality control: | None | ||||
Experiment variables
| Conditions | |
|---|---|
| · 500,000 NK cells were transfected with ONA/mRNA complex that contains 44.4 ng of NKG2D mRNA, 11.1 ng of DAP10 mRNA and 44.4 ng of GFP mRNA | 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B9 RP13_Unmixed.fcs |
| · 500,000 NK cells were transfected with ONA/mRNA complex that contains 42.9 ng of NKG2D mRNA, 14.3 ng of DAP10 mRNA and 42.9 ng of GFP mRNA. | 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C9 RP13_Unmixed.fcs |
| · 500,000 NK cells were transfected with ONA/mRNA complex that contains 40 ng of NKG2D mRNA, 20 ng of DAP10 mRNA and 40 ng of GFP mRNA. | 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D9 RP13_Unmixed.fcs |
| · 500,000 NK cells were transfected with ONA/mRNA complex that contains 50 ng of NKG2D mRNA and 50 ng of GFP mRNA. | 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A9 RP13_Unmixed.fcs |
| · 500,000 NK cells were transfected with ONA/mRNA complex that contains 100 ng GFP mRNA. | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E9 RP13_Unmixed.fcs |
| · NK cells were not transfected. | 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F9 RP13_Unmixed.fcs |
| Individuals | |
|---|---|
| · Donor RP44 (donor 1) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A2 RP44_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F2 RP44_Unmixed.fcs |
| · Donor RP45 (donor 2) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A3 RP45_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F3 RP45_Unmixed.fcs |
| · Donor RP47 (donor 3) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A4 RP47_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F4 RP47_Unmixed.fcs |
| · Donor RP48 (donor 4) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A5 RP48_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F5 RP48_Unmixed.fcs |
| · Donor RP49 (donor 5) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A6 RP49_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F6 RP49_Unmixed.fcs |
| · Donor RP50 (donor 6) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B7 RP50_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F7 RP50_Unmixed.fcs |
| · Donor RP07 (donor 7) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B8 RP07_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F8 RP07_Unmixed.fcs |
| · Donor RP13 (donor 8) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E9 RP13_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D9 RP13_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C9 RP13_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B9 RP13_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_GFP 1_1-A9 RP13_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F9 RP13_Unmixed.fcs |
| · Donor RP25 (donor 9) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B10 RP25_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F10 RP25_Unmixed.fcs |
| · Donor RP28 (donor 10) | 240123 NKG2D DAP10 and GFP co-transfection-Samples-GFP-E11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 2_1_2-D11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 3_1_3-C11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-NKG2D_DAP10_GFP 4_1_4-B11 RP28_Unmixed.fcs · 240123 NKG2D DAP10 and GFP co-transfection-Samples-Untransfected-F11 RP28_Unmixed.fcs |
