Freshly isolated or cultured hematopoietic populations (2–5×103 cells) were collected in complete media and plated on onto MicroWell 96-well glass-bottom plates (Thermo, Waltham, MA) coated with 1μg/mL poly-D-lysine or 30μg/cm2 Cell-Tak (Corning, Bedford, MA). Cells were allowed to adhere for 10min and fixed with 4% PFA/PHEM buffer for 15min. Cells were then permeabilized with 0.1% TritonX-100/PBS for 5min and blocked with 2% BSA/PBS for 1h at 4 °C. Cells were incubated with anti-pan-PMCA (1:250) overnight, washed and incubated with AlexaFluor secondary antibodies (Invitrogen) for 1h. Cell nuclei were counterstained with DAPI or DRAQ5 and mounted with fluorescent mounting media (Vector Labs, Burlingame, CA). Images were acquired with a Leica TCS SP8 confocal microscope or a Leica DMI 6000B and deconvoluted and processed with Leica AF6000 software package.
Microwell 96 well glass bottom plates
Manufactured by Thermo Fisher Scientific
MicroWell 96-well glass-bottom plates are high-quality, optically clear microplates designed for a variety of cell-based assays and microscopy applications. The plates feature a flat, thin glass bottom that provides excellent optical properties and compatibility with advanced imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dmi6000b, by Leica (3 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (3 mentions)
Fluorescent mounting media, by Vector Laboratories (3 mentions)
Af6000, by Leica (2 mentions)
Lsm 700 confocal microscope, by Leica (2 mentions)
Cell tak, by Corning (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
3 protocols using microwell 96 well glass bottom plates
1
Immunofluorescence Analysis of Hematopoietic Cells
2
Immunofluorescence Staining of Hematopoietic Cells
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Sorted or cultured hematopoietic populations (2–5×103 cells) were collected in complete media and plated on onto MicroWell 96-well glass-bottom plates (Thermo, Waltham, MA) coated with 1ug/mL poly-D-lysine. Cells were allowed to adhere for 10min and fixed with 4% PFA for 15min. Cells were then permeabilized with 0.1% TritonX-100/PBS for 5min and blocked with 2% BSA/PBS for 1h at 4 °C. Cells were incubated with anti-NFAT1 (1:100), anti-Mfn2 (1:200), anti-tubulin (1:200), anti CD150-APC (1:100) or anti-FLAG (1:250) (see Supplementary Table 1 ) overnight, washed and incubated with AlexaFluor secondary antibodies (Invitrogen) for 1h. Cell nuclei were counterstained with DAPI and mounted with fluorescent mounting media (Vector Labs, Burlingame, CA). Confocal images were acquired with a Zeiss LSM 700 confocal microscope or a Leica DMI 6000B and images were deconvoluted and processed with Leica AF6000 software package.
3
Immunofluorescence Staining of Hematopoietic Cells
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