Items Tagged ‘Fero-Flow’

June 3rd, 2016

Counts of Fluorescently Stained Cells

By UNM CC UNM CC

How to perform automated counts of fluorescently stained cells. M. Fero (7/2004) Note This protocol describes semi-automated cell counts using fluorescently labeled cells, a hemocytometer and ImageJ software. The hemocytometer is not needed if you have already calibrated the areas of images taken with your microscope and camera. Our setup automatically imports images in iPhoto […]

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June 3rd, 2016

96-Well Flow Setup Record

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Download the 96-Well Flow Setup Record

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June 3rd, 2016

Use of the B.D. FACS or Calibur Flow Cytometers

By UNM CC UNM CC

M. Fero — 12/02 Reservations Schedule time on a cytometer (FACS or Calibur) by filling in the calendar at the flow lab. Don’t grossly overbook since we have to pay for both reserved time and the time actually used (which ever is greater). Prepare Cells Cells should be stained ahead of time and suspended as […]

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June 3rd, 2016

Flow Sorting Fibroblasts with GFP and P.I.

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M. Fero — 3/02 Protocol Wash cells with PBS and trypsinize to a single cell suspension. Count an aliquot on a hemocytometer. Meanwhile centrifuge the cells (e.g. 1000 RPM, 5 min.) to pellet them. Resuspend the cells to a concentration of 2 x 106 cells/sample with Flow Sort solution. Aliquot into flow sort tubes (maximum […]

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June 3rd, 2016

Simultaneous Cell Surface and BrdU staining for Flow Cytometry

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Dan Kuppers 5/2011 (ref. Rothaeusler, K. and Baumgarth, N., Cytometry Part A 2006) Materials BrdU 10 mg/mL, store frozen 10 million in vivo labeled thymocytes PBS w/o Ca or Mg PBS w/ Ca/Mg (PBS with 0.1 g/L CaCl2, 0.1 g/L MgCl2•6H2O) Wash Solution: PBS + 0.5% NP40 Fixative: Fresh 1% paraformaldehyde + 0.05% NP40 Quick […]

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June 3rd, 2016

BrDU and PI Staining for Cell Cycle Analysis

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P. Olivier — 1/2000 Solutions 0.08% w/v Pepsin Dissolve 0.4 g pepsin in 500 mL of 0.1M HCl (496 ml dd H20 + 4.1 mL conc. HCl), filter, store 4ºC. 2M HCl For 417 mL ddH20 plus 83 mL conc. HCl. Filter, store RT. 0.1M Na Borate For 500 mL, 19.07 g Na borate, filter, […]

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June 3rd, 2016

Simultaneous Nuclear and Surface Immunostaining for Flow Cytometry

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M. Fero — 6/01 and D.B. — 12/98 Protocol Harvest 106 cells/sample with PBS/FBS into a 15 mL conical tube spin at 1300 RPM. Wash by resuspending cells in 1.5 mL of PBS/FBS by spinning at 3000 RPM in a microcentrifuge. Incubate with 100 µL (use Pharmacia antibodies at 1:500) of primary cell surface antibody […]

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June 3rd, 2016

Flow Cytometry of Fibroblast Nuclei for DNA Content

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S. Coats 1995 Materials P.I. Solution: 4 mM Na3Citrate (0.118 g/100 mL) 30 U/mL RNAseI (43 mg/100 mL) 0.1% Triton-X100 (0.1mL/100 mL) 50 µg/mL propidium iodide (5 mg/100 mL) Procedure Harvest cells: Rinse with a subconfluent 10 mL dish with PBS (Ca++/Mg++ free).  Cover with 1.5 mL 0.1% (2.5 mM) EDTA in PBS at 37ºC […]

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June 3rd, 2016

Lineage Analysis of Blood

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J. Orthel.  6/27/03 mf revised 9/04 Materials Capillary tubes 1.5 mL Eppendorf microfuge tubes 15 mL conical centrifuge tubes 96-well V-bottom plates (Corning Costar 3894, from Fisher) Flow tubes (Falcon 352054) Reagents 0.5 M EDTA pH 8.0 PBS + 0.5% heat inactivated FBS RBC Lysis Buffer 4.15 g NH4Cl 0.5 g NaHCO3 0.0186 g Disodium […]

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