Cell EM diagram citation, Lysosome / Example / 없는 것 table, mitochondria & aSyn notes, Sample preparation and process, Thawing and resting, Culture of PBMC, Vendors of PBMC table opening
Source: K. Kausch…R.A. Lichtman, J.T. Pichal, R.M. Levi, O.W. Press, L.J. Burns, M. Caligiuri, Williams Hematology, 9th edition, www.accessmedicine.com, Copyright © McGraw-Hill Education. All rights reserved.
| 있는 것 | Example | 없는 것 | |||
|---|---|---|---|---|---|
| ribosomes, occasional ribosome clusters, and strands of rough-surfaced endoplasmic reticulum (Fig. 73-2). Centrioles, mitochondria, microtubules (diameter approximately 0.25 μm), and microfilaments (diameter approximately 0.07 μm) are present in the cytoplasm adjacent to the cell membrane. The cytoplasm also contains lysosomes, which are approximately 0.4 μm in diameter, are electron opaque, and contain classic lysosomal enzymes (e.g., acid phosphatase, β-glucuronidase, and acid ribonuclease). | Golgi zone, are poorly developed | ||||
| GBA, GlcCer, GlcSph (Lysosome / 데anella) | 대략 종합하면, (Saskia thesis, and 뒤 summary), human PBMC에서 GBA activity, GBA protein 및 lysosomal markers 볼 수 있고, mouse에서는 GlcCer & GlcSph까지 볼수있음, 불명은 다음: GlcCer & GlcSph 변화자료 in human, BMP, Cathepsin D, LIMP2 |
LTI-291 clinical trial Ambroxol clinical trial: Lawson Health Re-search Institute (Atashrazm et al., 2018) | |||
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[Lysosome] - (anella - , 2018 #752) autophagosome, p62, autophagy-related genes (Atg5, LC3-II, Beclin-1). - (Alonzi, 2019 #757): LC3II measured and increased by CQ (by FlowCellect kit from Millipore) - (Papagiannakis, 2015 #753) HSC70, LAMP2 LIMP2, BMP? - Enzo's Lyso-ID® Red detection kit: quantifies delivery of LC3 into lysosomes, no culture, https://www.enzolifesciences.com/science-center/technotes/2012/june/directly-quantifying-autophagy-in-peripheral-blood-mononuclear-cells-pbmcs/ - (Ivanova, 2019 #759) 중요. ERT로 Partially normalized in GD PBMC (del Hoyo et al. 2010, PMID) mitochondira complex 등 잘 볼 수 있다네 | |||||
| Monocyte: The mitochondria are numerous, small, and elongated. |
aSyn gene expression 조금 하기는 함 https://www.proteinatlas.org/ENSG00000145335-SNCA/tissue, aSyn can also be isolated (HEO, 2004 #768), aSyn upregulated in PD | ||||
| Monocyte: The cytoplasmic granules resemble the small granules found in the granulocytic series, measuring approximately 0.05 to 0.2 μm in diameter. They are dense and homogeneous and are surrounded by a limiting membrane. These granules, as with the lysosomal granules of other leukocytes, are packaged by the Golgi apparatus after their enzymatic content has been produced by the ribosomal complex of the cell.10,11 These cytoplasmic granules contain acid phosphatase and arylsulfatase and, therefore, are primary lysosomes. After endocytosis, lysosomes fuse with the phagosome, forming secondary lysosomes. | (Bradshaw, 2020 #751, https://doi.org/10.1101/2020.02.12.945469. peer review certified 안 됬다고 pubmed 에는 이 논문 없어 PMID 없음) CCCP treatment led to MFN ubiquitination in primary fibroblasts, SH-SY5Y neuroblastoma cells and Jurkat leukaemic cells, treatment of PBMCs did not induce ubiquitination of MFN. PRKN mRNA and protein was readily detectable in PBMCs at comparable levels to those observed in Jurkat and fibroblast cells. In contrast, PINK1 protein was undetectable and PINK1 mRNA levels remarkably low in control PBMCs. Our findings suggest that the PINK1-Parkin mitophagy signalling pathway is not functional in PBMCs. Therefore, PBMCs are not a suitable biosample for analysis of mitophagy function in Parkinson disease patients. | ||||
| mouse | 2017 Saskia Thesis: in mouse PBMC, followings can be measured: GBA activity (table 2), GlcCer & GlcSph (table 3), Glycosyl-cholesterol (table S3), Gb3 (fig S3) | ||||
Sample preparation and process
- Blood draw → within 4 h → centrifugation → ship to Tsho (-4 vs frozen?). frozen is often used but not sure of functional decline.
- (good resource) PrecisionMed’s REVISED PBMC - Sample Handling Procedure 24 Hour incubation.pdf
Resources
- https://www.stemexpress.com/blogs/peripheral-blood-mononuclear-cells/
- https://oncohemakey.com/structure-receptors-and-functions-of-monocytes-and-macrophages/
Thawing and resting of PBMCs
- cPBMCs must be thawed carefully to avoid loss of cell viability and functionality. Samples are removed from liquid nitrogen and placed on ice, after which they are thawed in a 37°C water bath. Once there is a small crystal of ice left in the bottom on the tubes, 500 μL warm medium supplemented with 10% FBS, 1% penicillin/streptomycin, and L-glutamine is added drop-wise. The cells are then transferred to Falcon tubes containing 10 mL warm medium, and centrifuged for 5 minutes at 500 g to wash off the toxic DMSO. This wash step is repeated and the cell pellet is then resuspended in medium to count as before. Once thawed, PBMCs must be rested overnight to remove any apoptotic cells—this increases viability and improves functionality, and involves incubating the freshly thawed cPBMCs in supplemented medium for approximately 18 hours. After this resting period, cells must be washed, and are then ready to be used in culture or any other assay.
Culture of PBMC
- Protocol example
- {Bradshaw, 2020 #751}
- JS: They normally don’t proliferate in cultures, but do so when activated (to induce proliferation, phytohemagglutinin (PHA) or lipopolysaccharide (LPS) can be added to the culture medium at a concentration of 1-5 μg/mL)
- Because PBMCs comprise both lymphocytes and monocytes, some cells will adhere to the plate (monocytes/macrophages) while others may be in suspension (lymphocytes).
Vendors of PBMC
| BioIVT | PrecisionMed | Bay Biosciences | Sanguine | MJF | ||
|---|---|---|---|---|---|---|
| Inventory |
PD: One 이거안삼! Clinical site: Only one (Case ID 126787) a. No Hematologic or Oncologic conditions b. No Diabetic Conditions c. No Autoimmune disorders d. No Renal or Liver impairment e. No Dementia or other significant Mental illness | Absence confirmed | Absence confirmed (note) we do have PD serum and plasma samples available in stock. | Absence confirmed | To Proposal: include Fan, i) Nilo-PD is only advanced PD and ii) no HC. So PPMI is the ony option, having both HC and PD PBMCs. → they should require prelim data. | |
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Donor site: 411명 questionnaire DHQ, (Medication Deferral List, No cancer, leukemia, heart, lung, blood disease, infection, aspirin) Price 알려달라했음. | ||||||
| Ordered 202304 |
- PD donor: $240.00 x 6 vials x 1 donor = $1,440.00 - HC: $110.00 x 6 vials x 6 donors = $3,960.00 Total: $5400 | |||||
| prospective |
) to answer below: • Number of cases • Case criteria • Associated data required • Target number of cells per subject • Concentration per vial • Anticoagulant • Format (fresh or frozen) • Cryo-preservative (Frozen) (Fresh) • Storage Media • Special instructions Cell recoveries are variable and with disease state samples this can be more pronounced. Please supply inclusion/exclusion criteria for the enrollment of patients to increase cell yield and/or viability. Feasibility is contingent upon the requirements specified. For optimal results we recommend using K2 EDTA, or Iternatively Na citrate for the anticoagulant, and that the cell concentration be no more than 25M cell/vial. For media we recommend CryoStor CS10. |
feasibility for a custom collection, can you fill out the attached form → submitted (20230327), PBMCs from HC & PD (n=10 each group), ≥1 x 10^6 cells per case. → 7-9m, $84,000 (initial $21,000) → Js: I will contact you later |
? What would be the volume/number of cells per sample? Please provide more details of your requirements.--> Thank you for reply. We may revisit/refine our details of requirement before we finally decide but would like to find out the feasibility of sample acquirement with a preliminary details. • PBMCs from HC vs PD (n=~5-10 each), ≥1 x 10^6 cells per case. I assume getting the samples from healthy donors would be relatively easier vs PD patients. 소요시간 묻사 (2mil 으로) |
Meeting 20230322: to take 6-8m for 20 samples (1m for set up), to define exclusion criteria (to check Selnoflast protocol), 1m cells/ml, 100 m cells/pt, 100 ml blood/pt, cost: PD $3,000-4000/visit, HC: $2,000/visit → Js: I will contact you later, disease specific leukopaks from our patient population. These leukopaks could have 4-5 billion pbmcs for a half up to 8-10 billion pbmcs for a full. We can process these for you as well into aliquots of 50-100m cells/vial. | NA | |
| Paper work | MTA | Not necessary | ||||
| MSA | O (Confidentiality clause O, Material clause O) | O | ||||
| CDA | Not necessary | O | ||||
| ICF | O | |||||
| IRB | O | |||||
| SRI | ||||||
| Ariba registr | O | O | ||||
| Scientist.com | ||||||
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| top table / Lysosome row | (Lysosome / 데anella) | the bullet (anella reads as written; combined with the preceding (Lysosome] may be a single phrase Lysosome / organelle; preserved verbatim. |
| Vendors / Bay Biosciences cell | 소요시간 묻사 (2mil 으로) | reads as written; 묻사 could be a Korean shorthand of 문의 or 물어봤음; preserved verbatim. |