Immunofluorescence Microscopy of Neural Cells Differentiated from Peripheral Blood-Derived Cells

Abstract

Source: Becker-Kojić, Z. A., et. al., GM-Free Generation of Blood-Derived Neuronal Cells. J. Vis. Exp. (2021)

The video demonstrates a protocol for preparing neural cells derived from reprogrammed peripheral blood cells for fluorescence microscopy, showcasing distinct cell types and developmental stages through immunofluorescence imaging.

Protocol

1. Immunofluorescence microscopy analysis of blood-derived neural cells
   1. Culture the cells for 16 days and remove the media.
      1. Incubate with pre-warmed fixative consisting of 75 mL of sterile water, 4 g of paraformaldehyde. Add 10 N NaOH as needed and stir until the solution clears. Then add 10 mL of 10x PBS, 0.5 mL of MgCl₂, 2 mL of 0.5 M EGTA, and 4 g of sucrose. Titrate to pH 7.4 with 6 N HCl, and bring to 100 mL of sterile water for 15 min.
      2. Discard the fixative and wash the cells 3 times for 5 min each time. Immediately add a freshly made 0.3% Triton X-solution and permeabilize the cells for 5 min. Wash 3 times with PBS and add a blocking solution made by PBS and 5% BSA.
      3. Block the cells at room temperature on a rocker plate for 1 hour.
      4. Prepare appropriate dilutions of antibodies in 1% BSA/PBS and incubate the cells with antibody dilutions on a rocker plate for 1.5 h at room temperature. Wash the cells 3 times with PBS for 5 min each, incubate the cells with DAPI, and mount the coverslips with mounting media for visualization on a microscope.
         NOTE: Directly labeled antibodies used in this experiment are listed in the Table of Materials.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-GFAP Cy3 conjugate	Merck Millipore	MAB3402C3
Anti-MAP2 Alexa Fluor 555	Merck Millipore	MAB3418A5
Anti-Nestin Alexa Fluor 488	Merck Millipore	MAB5326A4
Anti-Tuj1 Alexa Fluor 488	BD Pharmingen	560381
BSA Frac V 7.5%	Gibco	15260037
Multiplatte Nunclon 4 wells	Fisher Scientific	10507591
Neurobasal Medium	Gibco	10888022
PBS sterile	Roth	9143.2
Poly-L-ornithine	Sigma-Aldrich	P4957-50ML