Sep 01, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.36wgq7xj5vk5/v1
  • 1Department of Biology, Wake Forest University;
  • 2Department of Microbiology & Immunology, Wake Forest School of Medicine
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DOI: dx.doi.org/10.17504/protocols.io.36wgq7xj5vk5/v1
Protocol Citation: Heather N Colvin, Glen Marrs, Regina Joice Cordy 2022. Osmolality-controlled fixation and simple preparation of human red blood cells for scanning electron microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7xj5vk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working.
Created: August 02, 2022
Last Modified: September 01, 2022
Protocol Integer ID: 68090
Keywords: erythrocyte, red blood cell, echinocytosis, scanning electron microscopy, osmolality, microscopy, fixation, human
Funders Acknowledgement:
NIH
Grant ID: K01HL143112
NIGMS
Grant ID: T32 GM127261
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.
Materials
ReagentPBS pH 7.4Thermo Fisher ScientificCatalog #10010023
ReagentDistilled WaterThermo FisherCatalog #15230162
ReagentGlutaraldehyde solution (50% in solution)Sigma AldrichCatalog #G6403
ReagentRound Glass Coverslips 12 mm diameterThermo Fisher ScientificCatalog #50-143-822
Safety warnings
Human blood carries a risk of possible transmission of bloodborne pathogens. Proper personal protective equipment (PPE) in accordance with biosafety level 2 research should be used to minimize this risk.

Glutaraldehyde should be used in chemical fume hood and disposed of according to institutional guidelines.


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

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 Amount300 µL 1X PBS (pH 7.4) + Amount200 µL 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)

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 Amount25 µL of packed, washed RBCs to Amount500 µL osmolality-controlled buffer from Step #1

  • Mix well with pipette, gently
Prepare osmolality-controlled 2% glutaraldehyde solution
Safety information
Caution: Prepare glutaraldehyde solution in chemical fume hood

  • CombineAmount240 µL dH2O, Amount240 µL 1X PBS (pH 7.4), Amount20 µL 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)



Fix RBCs with osmolality-controlled glutarldehyde

  • Add Amount500 µL osmolality-controlled 2% glutaraldehyde solution from Step #3 slowly to well-mixed RBC suspension from Step #2.
  • Invert gently in microcentrifuge tube
  • Incubate at TemperatureRoom temperature in the dark for Duration00:30:00
Note
Final osmolality of fixing solution ≈ 260 mmol/kg

Note: Osmolality measured with a Wescor VAPRO 5520 (vapor pressure osmometer)

30m
Wash glutaraldehyde-fixed RBCs

  • Centrifuge fixed RBCs after completion of Step #4 at Centrifigation900 x g, 00:05:00 , reduced acceleration and braking (Acc: 4/9, Dec: 2/9)
  • Remove supernatant without disturbing the RBC pellet
Safety information
Properly dispose of supernatant containing glutaraldehyde per institutional guidelines

  • Add Amount1 mL dH2O and mix pellet well with pipette
  • Centrifuge cells again Centrifigation900 x g, 00:05:00 , reduced acceleration and braking (Acc: 4/9, Dec: 2/9)
  • Remove supernatant without disturbing the RBC pellet




10m
Dilute fixed, washed RBCs in dH2O
  • Mix RBC pellet well with pipette and add Amount5 µL of fixed RBC pellet to Amount995 µL dH2O to create at 0.5% hematocrit (hct) suspension of washed and glutaraldehyde-fixed RBCs in water
  • Mix well and add Amount100 µL 0.5% hct glutaraldehyde-fixed RBC suspension to12mm 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 Temperature60 °C DurationOvernight



30m