Date: Wed Jun 22 2005 - 14:52:56 EST
<Bunny Cotleur wrote> I'm looking for advice from someone/anyone who is currently analyzing small cell numbers by flow cytometry. (NOT low frequency, but very few events to begin with). We are planning to start analyzing pediatric CSF samples in the near future. Since we are expecting very SMALL sample volumes with very FEW cells, I'm looking for advice on the best way to simultaneously quantify chemokine receptors, cytokine expression and cell phenotypes. Is flow cytometry the best method in this case? With very few TOTAL cells, should we try a microscopy approach? Can someone point (or SHOVE) me towards the best method? Our current equipment choices: At the moment, our Flow core is only equipped with a Facscan and a LSR1. I could feasibly do 4 color (with Viability on a 5th detector, using UV). We are waiting to find out if we will be funded for an Aria. In any case, we could actually take samples across town to be run on an Aria. (I'm thinking in terms of maximizing # colors at this point). We will also have a small amt of equipment money- enough for a FacsArray-type cytometer. Is there an advantage (as far as running smaller samples) vs the traditional continuous stream? (We've removed the droplet containment on the LSR1 years ago, so we already fixed the problem with the DCM sucking the sample before we get the bar pulled over. I regularly run 100ul samples without a problem). Our institution also has a well equipped Confocal /Fluorescent Microscopy core. Thanks for any input! bunny Laser scanning cytometry (LSC) is certainly the best instrumentation for samples with paucity of cells. Dr. Richard Clatch published several papers (see below) using LSC, reporting excellent results. He has the best experience in this area. Zbigniew Darzynkiewicz, M.D., Ph.D. Brander Cancer Research Institute at NYMC 19 Bradhurst Avenue, Suite 2400 Hawthorne, N.Y. 10532 darzynk@nymc.edu : Clatch RJ. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=11070846&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=11070846> Links <javascript:PopUpMenu2_Set(Menu11070846);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=11070846&itool=iconnoabstr&query_hl=6> Immunophenotyping of hematological malignancies by laser scanning cytometry. Methods Cell Biol. 2001;64:313-42. No abstract available. PMID: 11070846 [PubMed - indexed for MEDLINE] 2: Clatch RJ, Foreman JR. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9511939&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=9511939> Links <javascript:PopUpMenu2_Set(Menu9511939);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9511939&itool=iconabstr&query_hl=6> Five-color immunophenotyping plus DNA content analysis by laser scanning cytometry. Cytometry. 1998 Feb 15;34(1):36-8. Review. PMID: 9511939 [PubMed - indexed for MEDLINE] 3: Clatch RJ, Foreman JR, Walloch JL. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9511936&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=9511936> Links <javascript:PopUpMenu2_Set(Menu9511936);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9511936&itool=iconabstr&query_hl=6> Simplified immunophenotypic analysis by laser scanning cytometry. Cytometry. 1998 Feb 15;34(1):3-16. Review. PMID: 9511936 [PubMed - indexed for MEDLINE] 4: Clatch RJ, Walloch JL, Foreman JR, Kamentsky LA. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9199623&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=9199623> Links <javascript:PopUpMenu2_Set(Menu9199623);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9199623&itool=iconabstr&query_hl=6> Multiparameter analysis of DNA content and cytokeratin expression in breast carcinoma by laser scanning cytometry. Arch Pathol Lab Med. 1997 Jun;121(6):585-92. PMID: 9199623 [PubMed - indexed for MEDLINE] 5: Clatch RJ, Walloch JL. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9022735&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=9022735> Links <javascript:PopUpMenu2_Set(Menu9022735);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=9022735&itool=iconabstr&query_hl=6> Multiparameter immunophenotypic analysis of fine needle aspiration biopsies and other hematologic specimens by laser scanning cytometry. Acta Cytol. 1997 Jan-Feb;41(1):109-22. PMID: 9022735 [PubMed - indexed for MEDLINE] 6: Sinha A, Clatch RJ, Stuck G, Blumenthal SA, Patel SA. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=8931426&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=8931426> Links <javascript:PopUpMenu2_Set(Menu8931426);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=8931426&itool=iconabstr&query_hl=6> Isoflurane hepatotoxicity: a case report and review of the literature. Am J Gastroenterol. 1996 Nov;91(11):2406-9. Review. PMID: 8931426 [PubMed - indexed for MEDLINE] 7: Clatch RJ, Walloch JL, Zutter MM, Kamentsky LA. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=8659450&query_hl=6> Related Articles, <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Display&dop t=pubmed_pubmed&from_uid=8659450> Links <javascript:PopUpMenu2_Set(Menu8659450);> <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&do pt=Abstract&list_uids=8659450&itool=iconabstr&query_hl=6> Immunophenotypic analysis of hematologic malignancy by laser scanning cytometry. Am J Clin Pathol. 1996 Jun;105(6):744-55. PMID: 8659450 [PubMed - indexed for MEDLINE] -----Original Message----- From: bunny [mailto:bunny@cotleur.com] Sent: Tuesday, June 21, 2005 5:14 PM To: cyto-inbox Subject: Looking for advice on pediatric samples Bunny Cotleur, M.S. Sr Research Technologist, Dept of Neurosciences Scientific Consultant, Flow Cytometry Core Cleveland Clinic Foundation Lerner Research Institute, NC30 9500 Euclid Avenue Cleveland, OH 44195 Lab: (216)444-1164 ********************************************* Caminante, no hay camino. Se hace camino al andar.
