Hi Saraswathi, Spherotech and Bangs Labs have beads similar to Trucount beads, but not lyophilized in tubes and with all kinds of sizes. Check those catalogs to see if there's anything that catches your fancy. Also, a lot of this decision hinges on the device you're using. Bead population overlap in FSC/SSC plots are perfectly acceptable if you can get their fluorescence is in a channel unused for your actual stains - assuming you've got any left. Less evident but sometimes doable is to use a single channel for both beads as well as a stain - the beads will give you a nicely defined, high intensity peak at the same channel all the time, and if your most positive staining intensity in that channel is lower than that of the beads you can multiplex beads plus stain in that channel. Just make sure to use markers/gates to exclude that peak from reports on %positive population, MFI calculations etc. Best of luck, Guy <http://www.ablynx.com> Next generation therapeutic antibodies Guy Hermans, PhD Project Leader Ablynx NV Technologiepark 4 B-9052 Zwijnaarde Belgium <mailto:guy.hermans@ablynx.com> guy.hermans@ablynx.com tel: fax: mobile: +32 (0)9 261 06 57 +32 (0)9 261 06 27 +32 (0)486 788 551 <https://www.plaxo.com/add_me?u=30065269879&v0=994426&k0=2009290972> Add me to your address book... <http://www.plaxo.com/signature> Want a signature like this? -----Original Message----- From: Naravula, Saraswathi [mailto:saraswathi.naravula@dnax.org] Sent: Saturday, June 04, 2005 2:22 AM To: cyto-inbox Subject: Re: Right sized beads for 4-color human FACS panel without CD45 ( for absolute counts) Dear Flowers, I am doing a long term study to get absolute counts of cells with different markers on human PBMCs (4-color panel) using beads and would like to have a suitable sized beads so that I capture the beads on Forward versus Side Scatter without overlap/intereference with PBMCs which includes lymphocytes, monocytes and granulocytes. Also they have to be highly fluorescent (found in higher fluorescence channels than test fluorescent channels). One of the issue I am facing is high debris in lyse-no-wash Whole Blood (WB) procedure making files huge and when thresholded the smaller sized beads (I used Trucount beads, Size 4.2uM) are also thresholded. Without thresholding, due to enormous debris, the files become huge and un-analyzable. Could any of you suggest the right size beads and the vendor, probably bigger sized falling in high forward scatter region (at lymph/monocyte SSC level) without compromising on the LYMPHOCYTE /MONOCYTE/ GRANULOCYTE resolution/separation.(This helps me in better PBMC primary gating that I follow) Or could any of you suggest any other way of doing absolute counts from whole blood with beads without major issues. I appreciate your help. Saraswathi Naravula Scientist II, Dept of Discovery Research 901 California Avenue Palo Alto, CA 94304-1104 Ph: 650-496-6550 Fax: 650-496-1200 saraswathi.naravula@dnax.org ********************************************************************* This message and any attachments are solely for the intended recipient. If you are not the intended recipient, disclosure, copying, use or distribution of the information included in this message is prohibited -- Please immediately and permanently delete.
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