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H/M Pluripotent Stem Cell Multi-Color Flow Cytometry Kit

Key Product Details

Verification of Human BG01V Embryonic Stem Cell Pluripotency by Multi-Color Flow Cytometry.
(2)
Verification of Mouse D3 Embryonic Stem Cell Pluripotency by Multi-Color Flow Cytometry.
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FMC001

Assay Procedure

Assay Procedure

>Refer to the product datasheet for complete product details.

Briefly, stem cell pluripotency is assessed by flow cytometry using the following procedure:

  • Fix and permeabilize cells using Fixation/Permeabilization Buffer
  • Stain cells with fluorochrome-conjugated antibodies or isotype controls
  • Wash and resuspend cells in PBS
  • Analyze samples by flow cytometry
 

Reagents Provided

Reagents Supplied in the Human/Mouse Embryonic Stem Cell Multi-Color Flow Cytometry Kit (Catalog # FMC001)

  • SOX2-PE (Clone 245610; mouse IgG2A)
  • Oct-3/4-APC (Clone 240408; rat IgG2B)
  • SSEA-1-PerCP (Clone MC-480; mouse IgM)
  • SSEA-4-CFS (Clone MC-813-70; mouse IgG3)
  • Mouse IgM Isotype Control
  • Mouse IgG3 Isotype Control
  • Rat IgG2B Isotype Control
  • Mouse IgG2A Isotype Control
  • Fixation/Permeabilization Buffer (with 1% formaldehyde, 8.9% saponin, and <0.05% sodium azide)
  • Permeabilization/Wash Buffer (with 10% saponin and 0.05% sodium azide)
  • Includes enough reagents to perform 25 assays

Other Supplies Required

  • PBS or Hanks’ Balanced Salt Solution (HBSS)

Procedure Overview

Intracellular Staining Protocol with Simultaneous Fixation/Permeabilization

  1. Harvest cells and wash 2 times with PBS or HBSS.
wash with PBS or HBSS
  1. Resuspend cells in Fixation/Permeabilization Buffer (approximately 5 x 105 cells/0.5 mL) and transfer to 5 mL flow cytometry tubes.
  2. Incubate cells at room temperature for 30 minutes, vortexing intermittently.
resuspend cells in buffer and transfer to flow cytometry tubes
  1. Centrifuge samples at 300 x g for 5 minutes.
  2. Resuspend the pellet in 100-200 μL of Permeabilization/Wash Buffer.
resuspend the pellet in wash buffer
  1. Add 10 µL of each fluorochrome-conjugated antibody or the corresponding isotype control antibody.
  2. Incubate the samples at room temperature for 30-40 minutes in the dark.
add fluorchorome-conjugated antibody or isotype control antibody
  1. Wash the cells in Permeabilization/Wash Buffer.
  2. Resuspend the cells in 200-400 μL of PBS.
wash cells in buffer and resuspend cells in PBS
  1. Analyze the expression of pluripotent markers simultaneously by flow cytometry.
Analyze the expression of pluripotent markers simultaneously by flow cytometry

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