why wash cells with pbs before trypsin

DOT1L regulates chromatin reorganization and gene expression during Enrichment analysis showed overrepresented proteins that were associated to organelle, nucleus, and mitochondrion, suggesting cell burst after the PBS washing step. Method A Count the number of cells in the 4 outer squares (see the left panel of Figure 2). Unlike water, PBS prevents cells rupturing or shrivelling up due to osmosis. HBSS is designed for use with cells maintained in non-CO2 atmospheric conditions. A minimum of two, 25 cm2 flasks should be carried for each cell line; these cells should be expanded as necessary for the transfection experiments. Remove the wash solution. Wash the cells 3 times by centrifugation at 1500 rpm for 5 minutes and resuspend them in 200 l to 1ml of ice cold FACS buffer*. In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. Why do you wash cells in PBS? - Answers Careers, Unable to load your collection due to an error. International Journal of Molecular Sciences, http://creativecommons.org/licenses/by/4.0/, Stable isotope labeling with amino acids in cell culture. Either mix cells with an equal volume of trypan blue [0.4% (w/v) trypan blue in PBS] to determine live/dead count (dead cells are blue) or kill cells with 10% formalin and then stain with trypan blue or other another stain (to improve visualization of all cells). Previous question Next question. Be able to aspirate old feeding media from cell cultures, wash cells and feed cells with fresh media. To assess cell viability, cells were detached from plates by washing with 1 trypsin-EDTA at 37 C for 10 min. This step will require optimization. Akkk C.A., Liseth K., Hervig T., Ryningen A., Bruserud ., Ersvaer E. Use of different DMSO concentrations for cryopreservation of autologous peripheral blood stem cell grafts does not have any major impact on levels of leukocyte- and platelet-derived soluble mediators. The cells were permeabilized with PBS containing 0.3% Triton X-100 and 2% goat serum for 1 h, and they were subsequently incubated with goat anti-human FIX polyclonal . The saline solution is a solution of salt and All solutions and equipment that come in contact with the cells must be sterile. Aspirate the blocking buffer, and incubate the cells with the modified antibody solution according to the . The plasmid cocktail and lipofectamine were mixed and incubated at room temperature for 15 min before being added to a mixture of MDCK and HEK293 cells. Visually inspect cells frequently. Picotti P., Aebersold R. Selected reaction monitoring-based proteomics: Workflows, potential, pitfalls and future directions. Be able to appropriately thaw frozen cells and use specialized freezing media and cryopreservation vials to freeze cells. Live cells are phase bright; suspension cells are typically rounded and somewhat symmetrical; adherent cells will form projections when they attach to the growth surface. Adherent cultures that do not need to be divided can simply be fed by removing the old medium and replacing it with fresh medium. Thoroughly wash cell pellets with PBS before proteins are extracted. Zhang, Z. et al. For 5 x 106 - 3 x 107 cells use 2.0 ml RLT, up to 1 x Physiological parameters temperature 37C for cells from homeotherms pH 7.2-7.5 and osmolality of medium must be maintained humidity is required gas phase bicarbonate concentration and CO2 tension in equilibrium visible light, can have an adverse effect on cells; light induced production of toxic compounds can occur in some media; cells should be cultured in the dark and exposed to room light as little as possible. Alvites RD, Branquinho MV, Caseiro AR, Amorim I, Santos Pedrosa S, Rma A, Faria F, Porto B, Oliveira C, Teixeira P, Magalhes R, Geuna S, Varejo ASP, Maurcio AC. The manual calculation of the enrichment fold of the WebGestalt results is showed with an example (all proteins refers to the proteins uploaded in WebGestalt): Regulated proteins were imported into StringDB software version 10.5 for the analysis of protein interactions [33]. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca 2+ /Mg 2+. Key plasmids, Cas9-T6N and Cas12a-T8N have been deposited at Addgene (plasmid ID, 199604199605). The cells will then go into exponential growth where they have the highest metabolic activity. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Remove the wash solution. Bring up to the initial volume with water. An additional PBS wash and trypsinization step were performed to remove any cell surface-bound Cas9-CPP protein before FACS sorting or further downstream analyses. pH to keep tissues, cells, and proteins intact during If there is concern that the protein of interest is not being completely extracted from insoluble material or aggregates, RIPA buffer may be more suitable as it contains ionic detergents that will more readily bring the proteins into solution. Can I measure NADPH without lysing my cells? Do not re-use thawed aliquots. Bound peptides were eluted within a 195 min run using a binary gradient with buffer A (0.1% FA in water) and buffer B (0.1% FA in ACN). Cox J., Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. This study demonstrates that PBS can be used for the extraction of spermatozoa and cells from swabs and that PBS does not affect subsequent DNA profiling. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPRCas9 for genome engineering. Limma powers differential expression analyses for RNA-sequencing and microarray studies. The STRING database in 2017: Quality-controlled protein-protein association networks, made broadly accessible. 1.0% NP-40 (Triton X-100 can be substituted for NP-40). The authors declare no conflict of interest. Trypsinization - an overview | ScienceDirect Topics Correspondence to To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95100C for 5 min. Purchase these through your usual distributor. EXPLANATION : - PBS i . This is Part b, Tissue Culture Methods, under the module topic,Cell Culture Techniques. Article When red cells were found, 2 mL of lysis solution (NH 4 Cl 0.2 M, KHCO 3 0.01 M, EDTA 1.2 10 4 M) was added and the mixture was allowed to stand for 5 min; the reaction was stopped with 12 mL of 1X PBS; the cells were centrifuged as described above, resuspended in 1 mL of PBS, and counted. Aaseb E., Opsahl J.A., Bjrlykke Y., Myhr K.M., Kroksveen A.C., Berven F.S. Nature Biotechnology J Forensic Leg Med. 2023 Mar 6;17(2):024102. doi: 10.1063/5.0131806. Do you guys wash cells with PBS before collecting cells? Why? SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. Ballell-Hosa L, Gonzlez-Mira E, Santana H, Morla-Folch J, Moreno-Masip M, Martnez-Prieto Y, Revuelta A, Di Mauro PP, Veciana J, Sala S, Ferrer-Tasies L, Ventosa N. Pharmaceutics. Aspirate the PBS and resuspend the cells in 5 mL Complete Media and transfer into a T25 flask. Approximately 0.5 g peptides were pre-concentrated and separated, as previously described [5]. Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at 106 107 cells/ml. Confluent monolayers are dissociated with Trypsin-EDTA (1, 59430) for experiments and passages. National Library of Medicine trypsin active. Ryningen A., Ersvaer E., yan A.M., Kalland K.H., Vintermyr O.K., Gjertsen B.T., Bruserud . Stress-induced in vitro apoptosis of native human acute myelogenous leukemia (AML) cells shows a wide variation between patients and is associated with low BCL-2:Bax ratio and low levels of heat shock protein 70 and 90. eCollection 2020. Liquid Chromatography (LC)-MS Analysis. & Wherry, E. J. HHS Vulnerability Disclosure, Help Add 2 ml 1X Trypsin/EDTA. A. Centrifugation. To enable access of the antibody to this portion it is necessary to unfold the protein, ie denature it. 54, 874884 (2022). 2. Dilute in water, 10 mg/mL. Add 1 ml of trypsin to each 35 mm dish. Zetsche, B. et al. (Approximately 1,000,000 cells for a T-75 flask, 20,000 cells/well for a 24 well plate, 3200 cells/well for a 96 well plate.). lyse due to osmosis. 2. As soon as you have enough cells, several vials should be frozen away and stored in liquid N2. Dilute in ethanol. The viability of cells can be observed visually using an inverted phase contrast microscope. Wipe media tube with 70% ethanol and place inside the hood. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. As a library, NLM provides access to scientific literature. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Med. Epub 2015 Aug 13. Suspension cultures. HHS Vulnerability Disclosure, Help Use 10-15 ml for T-25s, 25-35 ml for T-75s and 50-60 ml for T-150s. Article Kleinstiver, B. P. et al. rachel.boulay@gmail.com | Ph: (808) 692-0986 | Fax:(808) 692-1973, 2023 Center for Cardiovascular Research - Molecular Biology. Biotechnol. Confirm that cells are at least 80% confluent by microscopy. PDF RNA Isolation from Tissue Culture Cells - dlmp.uw.edu Chen, Z. et al. Incubate the culture vessel at room temperature for approximately 2 minutes. Multiplex Protein Imaging through PACIFIC: Photoactive Densities lower than 20% confluency inhibit growth. However, a 27C controlled environment is recommended. Nat. Disperse the medium by pipetting over the cell layer surface several times. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. Unauthorized use of these marks is strictly prohibited. Nature 543, 113117 (2017). Rebecca Wangen performed the experiments. 157, 195206 (2009). R.M.K. Google Scholar. antibiotics, although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants. Air-Liquid Interface Culture of Nasal and Tracheal Airway Epithelial Cells Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Phosphate buffered saline (PBS) is a non-toxic solution used in many biological laboratories. This video explains why, when and how to passage cells grown in both adherent and suspension cultures. Nat. Leave UV light on for at least 5 minutes. 12, 492499 (2011). 8600 Rockville Pike Why do you wash cells in PBS? The dilution you use will depend on the density of the cells and how quickly they divide, which only you can determine. pH to keep tissues, cells, and proteins intact during & Pellois, J. P. Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. b. Spin down cells, remove supernatant and replace with fresh medium. Subculture cells as described below before confluency is reached. Biotechnol. Owers R, Davidson G, McDonald A, Morgan R, O'Rourke P. Forensic Sci Int. This topic part has two sections:Content Tutorial & Animation. Rebecca Wangen, Elise Aaseb and Frode Selheim analyzed the data. Cell debris was gated out and 10000 events were acquired for analysis. Immunol. 988.96 KB; 20-04-20. conceived and developed the Peptide-Assisted Genome Editing (PAGE) approach and designed the research. If prisons are adherent, remove of single culture media, wash in PBS, include enough trypsin to cover the cells and incubates forward approximately 2 min in a 37C incubator. ISSN 1546-1696 (online) Frangoul, H. et al. Qin, K. et al. Aspirate spent culture media from the cell culture vessel. Licensee MDPI, Basel, Switzerland. 33.jpg. June, C. H., OConnor, R. S., Kawalekar, O. U., Ghassemi, S. & Milone, M. C. CAR T cell immunotherapy for human cancer. INTRODUCTION : - Cells are washed to remove extra serum, proteins, or unbound reagents with a physiological buffer solution during the cell culturing process and washing is also essential for the immunofluorescence procedures. Bruserud ., Gjertsen B.T., Foss B., Huang T.S. 1 Pt A. Verheggen K., Raeder H., Berven F.S., Martens L., Barsnes H., Vaudel M. Anatomy and evolution of database search engines-a central component of mass spectrometry based proteomic workflows. Expert Answer. DOC Protocol to Sub-culture ("Passage", "Pass", or "Split") Cells Frequent feeding is important for maintaining the pH balance of the medium and for eliminating waste products. Wipe incubator door and microscope stage with 70% ethanol. Cells should be monitored daily for morphology and growth characteristics, fed every 2 to 3 days, and subcultured when necessary. In general, all you need to do is aseptically combine several sterile solutions. Then the cells were fixed with ice-cold methanol for 15 min. Whenever cells are in suspend, just transfer the desired output directly inside a 50 mL Falcon tube. 3. Next day, transfer to liquid nitrogen (DONT FORGET) and log in the Liquid Nitrogen Freezer Log Book, if applicable. Mol. Andrea Trentani, ystein Bruserud and Maria Hernandez-Valladares contributed materials. Bethesda, MD 20894, Web Policies Science 354, 11601165 (2016). 3. When adherent cells become semi-confluent, subculture using 2 mM EDTA or trypsin/EDTA. Observe cells under the microscope and incubate until cells become rounded and loosen when flask is gently tapped with the side of the hand. Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. To obtain Trypsin should be . Counting procedure for Methods A (left panel) and B (right panel). Work in the Wherry lab is supported by the Parker Institute for Cancer Immunotherapy. Ideally, cells are harvested when they are in a semi- confluent state and are still in log phase. This video explains why, when and how to passage cells grown in both adherent and suspension cultures. Accessibility Note that the actual incubation time varies with the cell line used. Science 361, 285290 (2018). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (, GUID:10B2B901-69A9-40FA-B084-9C79052E814B, proteomics, acute myeloid leukemia, preservation, phosphate buffered saline, dimethyl sulfoxide, mass spectrometry, sample preparation. In general, mammalian cells should be handled gently. Drug Discov. Inclusion of 2-mercaptoethanol or dithiothreitol in the buffer reduces disulphide bridges, which is necessary for separation by size. Trypsinization Procedure - Lonza Bioscience Rev. 5. Restoring function in exhausted CD8 T cells during chronic viral infection. You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 min at 12,000 rpm but this must be determined for your experiment (e.g. Raw data were processed with MaxQuant version 1.5.5.1 [25,26]. What is sterile pbs? Place culture in the 37C incubator for 2 minutes. It is not the PBS that causes the confluent. PDF Cell culture guidelines - Abcam 19, 770788 (2018). 4. In principle, HNEpCs are easier to obtain than HBEpCs or HTEpCs, as they are isolated from the normal human nasal mucosa. Cell culture media has trypsin neutralizers, so when you wash Freezing or Recovering Cells - Cell Biologics Dilute cells with fresh medium and transfer to a sterile centrifuge tube. Therefore, it is essential to remove all traces of serum from the culture medium by washing the monolayer of cells with PBS without Ca2+/Mg2+. The GRCh38/hg38 human reference genome is publicly available. Resuspend cells in complete medium and determine cell count/viability. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. Aspirate the media, leaving a small layer of media on top of the cell pellet. Spin cells down, remove supernatant, and resuspend in culture medium (or freezing medium if cells are to be frozen). E. John Wherry, Shelley L. Berger or Junwei Shi. Nucleic acid detection with CRISPR-Cas13a/C2c2. Why is it necessary to wash adherent cell lines in PBS/DPBS before I normally wash the cells with PBS before adding trypsin (for 5 min). Phosphate Buffered Saline (PBS): a salty solution of The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Nature, 227, 6805). This topic part has one section:Content Tutorials. To test for sterility after adding all components, pipette several milliliters from each media bottle into a small sterile petri dish or culture tube and incubate at 37C for several days. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice. The https:// ensures that you are connecting to the Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. 1. Cells should only be exposed to trypsin/EDTA long enough to detach cells. Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Volumes of lysis buffer must be determined in relation to the amount of tissue present. Viability can also be assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells. Genet. Add PBS at a volume to deliver 10 10 6 cells in 0.1 ml, . It is also essential to keep your cells as happy as possible to maximize the efficiency of transformation. Pipette cell suspension into a 15 mL centrifuge tube. G.A.B. When adherent cells become semi-confluent, subculture using 2 mM EDTA or trypsin/EDTA. Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, USA, Zhen Zhang,Diqiu Ren,Kunhua Qin,Sierra M. Collins,Hua Huang,Chad A. Komar,Peter F. Bailer,Gerd A. Blobel,Rahul M. Kohli,Shelley L. Berger&Junwei Shi, Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA, Zhen Zhang,Sierra M. Collins,Hua Huang&Shelley L. Berger, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, USA, Amy E. Baxter,Zeyu Chen,Hua Huang&E. John Wherry, Institute for Immunology and Immune Health, University of Pennsylvania, Philadelphia, PA, USA, Amy E. Baxter,Zeyu Chen&E. John Wherry, Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA, USA, Division of Hematology, The Childrens Hospital of Philadelphia, Philadelphia, PA, USA, Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA, Peter F. Bailer,Jared B. Parker&Rahul M. Kohli, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA, Parker Institute for Cancer Immunotherapy, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, You can also search for this author in and J.S. & Kleinstiver, B. P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Aspirate off existing media from the flask or microplate. This method is quick and easy but is also disruptive to the cells and may result in significant cell death. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Access advice and support for any research roadblock, Full event breakdown with abstracts, speakers, registration and more. The standard loading buffer is called 2X Laemmli buffer (Laemmli UK, 1970. Incubate cells at 37C for approximately 5 minutes until cells are detached. After staining with primary antibody cells were washed in PBS and secondary antibody goat anti-mouse IgG-AlexaFLuor-555 (1:100, Life Technologies) were added and incubated for 1 hr at 4C. Measure out the desired amount of media and pipette into a centrifuge tube. Rule of thumb: reduce and denature unless the datasheet specifies otherwise. Wangen R., Aaseb E., Trentani A., Dskeland S.O., Bruserud ., Selheim F., Hernandez-Valladares M. The enrichment analysis of DMSO vs. pellet samples showed approximately the same results for biological processes and cellular components as for the DMSO vs. SDS samples. When culturing cells, and particularly for immunofluorescence procedures, cells are washed with a physiological buffer solution to remove extra serum, proteins, or unbound reagents. Observe the cells under the microscope for detachment. Caution: We do not recommend shaking the flask vigorously, because it may result in damage to the cells. Epub 2018 Apr 14. Most cells are passaged (or at least fed) three times a week. just as many ions per unit volume as the inside of a cell (so that Why do cells recovered from liquid nitrogen have lower viability. Nat. Maintain constant agitation for 30 min at 4C. Rev. 12, 19801998 (2017). All media preparation and other cell culture work must be performed in a laminar flow hood. In cases where it is important to preserve protein-protein interactions or to minimize denaturation, a buffer without ionic detergents (eg SDS) and ideally without non-ionic detergents (eg Triton X-100) should be used. Spin the cells down at 1 rcf x 5 min. is a member of the Parker Institute for Cancer Immunotherapy which supported this study. Do you have any idea of what is happening? Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. PubMedGoogle Scholar. Check cells under microscope and place in incubator. Google Scholar. sterilized (either by filter or by. In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer. Before Most of the media components will be purchased prepared and sterile. SDS grade is important for high-quality protein separation: a protein stained background along individual gel tracts with indistinct or slightly distinct protein bands are indicative of old or poor quality SDS. Pharmaceutics | Free Full-Text | Internalization and Transport of acknowledges NIH/NHLBI (R01-HL119479). A universal preservation method of the AML samples cannot be selected for all subsequent cell-based, genomics and proteomics studies. When you failed to wash the cell monolayer with PBS/DPBS, the action of trypsin was inhibited by serum traces present in the cell monolayer. Glutamine. Examine the cells to ensure the cells are healthy and free of contamination Remove and discard the culture media from flask Gently rinse the cells with balanced salt solution without Ca +2 and Mg +2 ions and remove the solution. cells. trypsin active. As soon as cells are in suspension, immediately add culture medium containing serum. Wash cells once with serum containing medium and dilute as appropriate (generally 4-20 fold). Remove and discard the spent cell culture media from the culture vessel. with PBS beforehand you are washing away these factors, making Cell 157, 12621278 (2014). The scratched cells were washed with PBS, and the scratch width was photographed with an inverted microscope at 0 h and measured with Image J software. Milone, M. C. et al. PDF 1. Subculture Materials Procedures - University of California, San Diego 15, 486499 (2015). Dilute in water. 3 Item(s) J.S. Tris-Triton buffer (cytoskeletal proteins). Before desalting, the extracts were acidified with 1% formic acid. The recommended split ratio for primary murine cells is 1:2. Clement, K. et al. Z.Z., A.E.B., Z.C., J.B.P., R.M.K., E.J.W., S.L.B. The minimum recommended concentration is 0.1 mg/mL, optimal concentration is 15 mg/mL). Dobin, A. et al. Pipette 6 ml of media into flask and pipette up and down against cell wall until cells have become suspended. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Z.Z., E.J.W., S.L.B. The site is secure. 3. Int J Cell Biol. Wu, Y. et al. Why is PBS used to wash cells before trypsin? 37, 224226 (2019). Insect cells attach very tightly to substrates under serum-free conditions and require additional effort to detach. This can be useful when trying to obtain a signal for a weakly-expressed protein. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. (in press). Release of angiopoietin-1 by primary human acute myelogenous leukemia cells is associated with mutations of nucleophosmin, increased by bone marrow stromal cells and possibly antagonized by high systemic angiopoietin-2 levels. Weissman, I. L. & Shizuru, J. what makes muscle tissue different from other tissues? 8600 Rockville Pike Wipe centrifuge tube with 70% ethanol and place back into the hood. Cells should only be exposed to trypsin/EDTA long enough to detach cells. The proteomics quality control software PTXQC was used to check LC-MS data quality from output files generated by MaxQuant [28]. Bethesda, MD 20894, Web Policies 2. Nat. In this topic youll learn about the role of Maintaining Cells. Tap bottom of flask lightly to detach more cells. You may also tap the vessel to expedite cell detachment. Why do we use PBS for washing? - Heimduo official website and that any information you provide is encrypted Explain why we have to wash the cells with PBS first before using Trypsin, what would be the possible effect of having 1. Cox J., Neuhauser N., Michalski A., Scheltema R.A., Olsen J.V., Mann M. Andromeda: A peptide search engine integrated into the MaxQuant environment. Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs. Trypsin-EDTA : University of Calgary Reference, P.J. Sample Preparation | Proteomics and Metabolomics (PMC) - UTHSC PDF Special Techniques Cell Pellet Protocol - National Institute of

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