| Sporadic PD | NIIDS | NID27600 | M | 69 | 65 | 52 | Caucasian | absent | ? |
| 실험표 등 cs- 00201270 | NIIDS | NID25202 | M | 60 | 59 | 59 | Caucasian | absent | ? |
| NIIDS | NID27132 | M | 66 | 60 | 60 | Caucasian | absent | ? | |
| NIIDS | NID30150 | F | 76 | 74 | 72 | Caucasian | absent | ? |
Budget
| ¥ | $ | ARIBA | ARIBA | SAP | |||
|---|---|---|---|---|---|---|---|
| Taka | Culture, MC1 functional assays, proteomics | ¥1,800,000 (one point eight million yen) | $16,000 (sixteen thousand usd) | Already transferred from NSTM-Shonan to PhReT | |||
| Radioligand binding assay | RI (FIRST BATCH) | ¥1M ?(Tomimatsu) | $2700 (줄서? | 못 찾겠다 함 (Tomimatsu 20220201) | I have put $100,000 in the BoB | ||
| TNTecnos consignment | <¥1M (Tomimatsu) | Should be cheap (cf. Charles river $30,000 per cell) | NA | The new (completed in Dec) study cost will appear in Jan. Culture ¥295,350 + binding assay ¥160,000 | |||
| sPD fibroblast purchase | ¥ 241,070 (cf. Nankai: ¥ 30,859 책상에 둔거) | $2,197.94 ($500 per cell): $2,000 (cells) + $197.94 (shipping | $2197.94 | PR745805 -8000550197 (PO겟지) Items received Jul 2021. | ¥ 241,070. This doesn't appear in SAP yet because payment in progress. | ||
| Another RI purchase | ¥1M정도라고 (Tomimatsu) | 환산하면 $23k - 31k | Invoice: -RI filter binding assay: ¥960,000, -細胞培養業務: ¥1,712,200 합: ¥2,672,200 (=~$20,010) | Araki대신구매에정 | |||
| Another TNTecnos consignment | the expansion of remaining 10 fibroblasts may take 2-3 m, total cost should be 3-4 million JPY | ||||||
| (tbd) human brain ARG | - Payment to Juntendo uni? - (radioligand, TNT는 공동) | ||||||
| MC1 PET Study | $400,000 (Paul McQuid budgeted) | QTS has budgeted for FY2021 (?) |
Sample preparation examples
| Reference | Sample | Fraction | Method | Purpose | Whole cell or mito-enriched |
|---|---|---|---|---|---|
| (Kazami, 2019 #1082) bovine heart | Bovine heart tissue | cardiomyocyte SMP (sub-mitochondrial particles) | 6. Mito isolation: {Smith, 1967 #1240} beef heart, grinding → homogenizing, fractionation (centrifugation) → 7. SMP preparation: {Thierbach, 1981 #1242} further sonication → centrifugation | in vitro radioligand binding assay | Mito-enriched fraction |
| (Zilocchi, 2020 #901) | Human fibroblast | Mito-enriched fraction | (commercial kit사용, sucrose언급x) Cell Lysis → fractionation (spin) (→ validation by WB, assessing VDAC1, CS, histone H3) | Proteomics | Whole cell pellet & mito-enriched fraction |
| (Basu, 1978 #1243) Paul McQuade | Human fibroblast | Plasma membrane | Culture → subculture → harvest (lysis, scraping) → spin → homogenizer → spin. | in vitro radioligand binding assay | |
| (Von Ruecker, 1984 #1245) | Human fibroblast | SMP | 1. Mito isolation (우리가 만들 mito-enriched sample이 이거일까?): Harvest (trypsination) → sucrose → homogenizing, fractionation (centrifugation) → 2. SMP preparation: further sonication → centrifugation | Enzyme assay |
Shipping
| Goods | Shipper (origin) | Description of goods | Dry ice | Destination address | Destination Consignee |
|---|---|---|---|---|---|
| Part 1 | 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan, Takeda Pharmaceutical Company Limited, | Sebastian Sjoqvists sebastian.sjoqvist@takeda.com +81466321351 | |||
| Part 2 | 9625 Towne Centre Dr, San Diego, CA, USA, 92121, Takeda, Neuroscience Biology | Paul Rolzin +1 619-930-8318 (office TCD.3800-P) paul.rolzin@takeda.com | |||
| Part 3 | Takeda Development Center Americas, Inc, 35 Landsdowne Street, Cambridge, MA 02139-4232, US 125 Binney Street 3.228/3.235 Cambridge MA Takeda 02142 USA | Chris Leonard Chris.leonard@takeda.com 6175518886 (Room 35-5052A) Ashley Knight (office 125BS) |
Sample preparation original plan
| Culture | MC1 enz assay | proteomics | Radioligand binding study | |||||
|---|---|---|---|---|---|---|---|---|
| 4 control & 4 Parkin-PD | 8 | 4 control & 4 Parkin-PD | 8 | 4 control & 4 Parkin-PD | 8 | 4 control & 4 Parkin-PD | 8 | |
| N=3 | 3 | N=3 | 3 | N=3 | 3 | N=3 | 3 | |
| Culture중 | Glucose & Galactose | 2 | Glucose & Galactose | 2 | Glucose & Galactose | 2 | ? Glucose & Galactose | 2 |
| harvest이후 | Whole cell & mito-enriched | 2 | Whole cell | 1 | Whole cell | 2 | ? Whole cell & mito-enriched | 2 |
| Total | 96 | 48 | 24 | |||||
20210326: passage number 8-12
Sample shipping : frozen cell vs sample
| Tbos에 뭘 보내나? 우측 셋 단계중 어디서 보내나? | Frozen cell, | Fibroblast 가능 {Von Ruecker, 1984 #1245} | Tbos Culture | Mito isolation |
| mio-enriched fraction ('Sample') | 가능? 일단 harvest후엔 shipping 아마 안 되겠지. Tbos에서 frozen받아서 녹여 culture해야겠지. 아니, 내가 정인바이오에 sample을 보낸경험, 결국 Tbos가 결정해야 함. | |||
| SMP ('Sample') | 가능? |
Update 20210423 (Taka)
| MC1 activity | No difference in glucose/galactose/CCCP-condition | |
| MC1-ATP coupling assay | No difference in glucose/galactose condition | |
| Caspase3/7 | ↑ trend | |
| MitoTrackerGreen (mitos)/DRAQ5 (nuc) | = | |
| SYBR green ((mt)DNA)/MitoTrackerRed (mitos) | ||
| Seahorse assay | = in glucose condition (No difference, rather higher maximum respiration |
samples for mitophagy and mito-membrane potential assays and CellPainting were collected but not performed the assays (ICC, imaging, quantification etc)
Update 20210806 (Yoshirou)
probe: JRAM
| homogenate | Living cells | Cell lysate | |||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Healthy (n=2, GM01650, GM03652) | Healthy (n=2, GM01650, GM03652) | ||||||||||||||||||||||||
| concentration | Protein | 10, 20, 50, 100, 200 ug | Not measured, but expected to be far lower than those in homogenate study | ||||||||||||||||||||||
| cell | Two (1 × 10⁴ cell/mL, 2 × 10⁴ cell/mL) | ||||||||||||||||||||||||
| 3H-BCPP-EF | 1 nM | Five (6.25, 12.5, 25, 50, 100 nM) | |||||||||||||||||||||||
| Rotenone block | 1 uM | 1 uM (too little?) | |||||||||||||||||||||||
| If block (cold probe) | Not done | Not done | Not done, easier, but not favorable if we add detergent) | ||||||||||||||||||||||
| protocol |
Culture in a large flask → Get homogenate Based on the calculation with assumption of efficiency in homogenize is 60%, 3*10^5 cells may provide 200 µg total protein.
|
Day1 Place the cells - 20,000 cells/100uL/well, over night incubation in CO2 incubator (37°C, 5%) Day2 Wash the cell with PBS 200uL/well once → Add 50 MEM with 2×DMSO or 2×Blocking compound → Add 50 MEM with 2× 3H-BCPP-EF: This results in 1X DMSO/compound with 1X 3H-probe
| |||||||||||||||||||||||
| result interpretation |
the clear S/B in this condition, with clear protein concentration-dependency. So the probe should work at least in homogenate
-%depletion should be <10%, (이 말은 less than 10% of the ligand binds 이라는 말은 아님) |
Ashley: counts are too low. we prefer to see counts around 1000-1500 dpm; which is approx. around 500 cpm on the low end; this is preference based on the typical limits of the scintillation counter). [bar chart: y-axis CPM 0-3000; x-axis Concentration (nM) 6.25, 12.5, 25, 50, 100; bars by group: Total binding (10K cells), Rotenone (10uM, 10K cells), Total binding (20K cells), Rotenone (10uM, 20K cells)] | |||||||||||||||||||||||
(continues on next photo: “…→ 대략 the free concentration of ligand is approximately equal to the concentration you added. → This assumption vastly simplifies the analysis of binding experiments, and the standard analysis methods depend on this assumption. → ligand depletion 이 10% 이하인…“)
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Top SNP demographic table left-most labels (Sporadic PD / 실험표 등 cs- 00201270) | “00201270” / sub-header label | Top edge of page is partially clipped; demographic-table sub-labels in Korean / numeric form transcribed as visible. |
| Budget row “Tomimatsu” notes “(Tomimatsu)“ | exact author tag | Several budget cells contain a parenthetical “(Tomimatsu)”; transcribed from the visible glyphs. |
| ARIBA column “PO겟지" | "PO겟지” | OCR shows “PO겟지” (i.e. “PO인 듯”); transcribed as seen. |
| Sample preparation original plan column count totals “96 / 48 / 24” | matrix totals | Final totals row appears partially clipped; “96” and “48” derived from displayed cells. |
| Update 20210806 protocol bar-chart legend wording | exact bar-group labels | Bar chart legend uses small abbreviated labels (“Total binding (10K cells)”, etc.); transcribed from the most legible reading. |
| Update 20210806 result interpretation right-most “Cell lysate” column body | empty / continuation | Right-most “Cell lysate” column has only a “Not done, easier, but not favorable if we add detergent)” cell; remaining cells are blank in this capture. |