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    Osmolality-controlled fixation and simple preparation of human red blood cells for scanning electron microscopy

    Heather N Colvin, Glen Marrs, Regina Joice Cordy

    Published: 2022-09-01 DOI: 10.17504/protocols.io.36wgq7xj5vk5/v1
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    Abstract

    Scanning electron microscopy (SEM) provides a way to visualize red blood cell (RBC) morphology. Previous methods for human RBC fixation can induce osmotic-related changes to healthy RBCs, which can interfere with interpretation of biological morphological changes. In addition, traditional methods for fixation and dehydration of RBCs and associated SEM preparations involve multiple chemicals and time-intensive steps. Here, we provide a simplified protocol for human RBC fixation with careful control of osmolality. This protocol omits the use of sodium cacodylate, osmium tetroxide, and ethanol gradient dehydration steps, yet results in comparable outcomes.

    Before start

    In order to obtain the human blood needed for these studies, ethical clearance must first be obtained.

    Steps

    1.

    Note
    Note the following protocol includes volumes to fix one (1) sample of packed, washed human red blood cells (RBCs). For multiple samples, scale up all volumes proportionately

    Make osmolality-controlled buffer

    • Add 300µL 1X PBS (pH 7.4) + 200µL distilled water (dH2O) in microcentrifuge tube for 500uL total osmolality-controlled buffer solution

    • Mix well

      Note
      Osmolality ≈ 160 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)

    2.

    Suspend human RBCs in osmolality-controlled buffer

    Safety information
    Caution: Institutional bloodborne pathogen biosafety precautions should be followed when working with specimens containing unfixed human blood

    • Add 25µL of packed, washed RBCs to 500µL osmolality-controlled buffer from Step #1

    • Mix well with pipette, gently

    3.

    Prepare osmolality-controlled 2% glutaraldehyde solution

    Safety information
    Caution: Prepare glutaraldehyde solution in chemical fume hood

    • Combine240µL dH2O, 240µL 1X PBS (pH 7.4), 20µL 50% glutaraldehyde in conical tube and mix well by inverting
      Note
      Osmolality ≈ 350 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)
    4.

    Fix RBCs with osmolality-controlled glutarldehyde

    • Add 500µL osmolality-controlled 2% glutaraldehyde solution from Step #3 slowly to well-mixed RBC suspension from Step #2.
    • Invert gently in microcentrifuge tube
    • Incubate at Room temperature in the dark for 0h 30m 0s
      Note
      Final osmolality of fixing solution ≈ 260 mmol/kg Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)
    5.

    Wash glutaraldehyde-fixed RBCs

    • Centrifuge fixed RBCs after completion of Step #4 at 900x g

    • Remove supernatant without disturbing the RBC pellet

      Safety information
      Properly dispose of supernatant containing glutaraldehyde per institutional guidelines

    • Add 1mL dH2O and mix pellet well with pipette

    • Centrifuge cells again 900x g

    • Remove supernatant without disturbing the RBC pellet

    6.

    Dilute fixed, washed RBCs in dH2O

    • Mix RBC pellet well with pipette and add 5µL of fixed RBC pellet to 995µL dH2O to create at 0.5% hematocrit (hct) suspension of washed and glutaraldehyde-fixed RBCs in water

    • Mix well and add 100µL0.5% hct glutaraldehyde-fixed RBC suspension to 12mm round Poly-L-Lysine coated coverslide* on slide warmer

      Note
      *Use the most appropriate slide size for the specific scanning electron microscope that will be used to image cells

    • Dry slides on slide warmer for 60°C``0h 30m 0s

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